Compositions and methods for treating neuropsychiatric disorders using an endothelin-b receptor agonist

ABSTRACT

The present invention relates to compositions and methods for treating neuropsychiatric disorders in vertebrates and humans. More specifically, the present invention provides for use of an endothelin-B receptor agonist as a neuroprotective and a neuroregenerative agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/903,574 filed Jan. 7, 2016, which is a U.S. National Phase ofInternational Application No. PCT/US2014/45748 filed Jul. 8, 2014, whichclaims the priority benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalPatent Application No. 61/843,702, filed Jul. 8, 2013, and U.S.Provisional Patent Application No. 61/902,935, filed Nov. 12, 2013, thedisclosures of which are incorporated herein by reference in theirentirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename: 48812_SeqListing.txt;created Jul. 8, 2014, 659 bytes—ASCII text file) which is incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatingneuropsychiatric disorders in vertebrates and humans. More specifically,the present invention provides for use of an endothelin-B receptoragonist as a neuroprotective and a neuroregenerative agent.

BACKGROUND OF THE INVENTION

Endothelin (ET) is an endogenous peptide which has been implicated innumerous physiological and pathological phenomena within the body.Acting upon two distinct receptors, ET_(A) and ET_(B), ET influences arange of processes from regulation of blood pressure toneurotransmitters and hormones (Kojima et al., 1992; Levin, 1995;Schiffrin et al., 1997; Schneider et al., 2007). Although most widelystudied for their actions on cardiovascular system, ET receptors arewidespread throughout the body, including the brain. ET_(B) receptors,specifically, are located in abundance on neurons and glial cells, aswell as endothelial lining of the cerebral vasculature (Schinelli,2006). The exact function of these receptors within the brain,particularly during its development, is not well understood.

Development of the Central Nervous System

A deficiency in ET_(B) receptors at birth has been shown to result in adecrease in neuronal progenitor cells and an increase in apoptosiswithin the postnatal dentate gyrus and cerebellum of rats (Ehrenreich etal., 2000; Vidovic et al., 2008). Additionally, ET_(B) knockout model inrats leads to congenital aganglionosis within the gut and associated CNSdisturbances (Dembowski et al., 2000). These ET_(B) knockout rats, whichhave a 4 week postnatal mortality, serve as models for humanHirschsprung disease. Previous studies have shown that brain ET_(B)receptor expression is particularly high immediately after birth, butdrops down to lower levels by postnatal day 21 (Briyal et al., 2012b).The locations of these receptors and their correlation, or lack thereof,with CNS growth factors during these crucial stages of developmentremain to be determined.

While it is clear that ET_(B) receptors are needed for normal CNSdevelopment, it remains uncertain which cells or pathways they exert aprotective or proliferative influence on. Previous studies have shownthat selective stimulation of ET_(B) receptors produces neuroprotectionagainst oxidative stress and a significant reduction in infarct volumein the brains of adult rats subjected to cerebral ischemia (Leonard etal., 2011; 2012). It was also found that protection and recovery fromthe ischemic condition was at least partially due to an increase inangiogenesis and neurogenesis within 7 days following infarct andtreatment with ET_(B) receptor agonist, IRL-1620 (Leonard and Gulati,2013). An increase in vascular and nerve growth factors within the brainof IRL-1620-treated infarcted animals coincided with an increase in thelevel of ET_(B) receptors.

Vascular endothelial growth factor (VEGF) is expressed normally in thecerebral microvessels as well as in the neuronal tissue of both neonatesand adults (Hoehn et al., 2002). VEGF in the fetal human brain islocated on neuroepithelial cells, neuroblasts, radial glial cells andendothelial cells, and its expression appears to be developmentallyregulated and correlated with angiogenesis (Virgintino et al., 2003).While VEGF is well known to be necessary for blood vessel growth, recentresearch has indicated that it also plays a significant role inpromoting neurogenesis, neuronal patterning, and neuronal migration(Rosenstein et al., 2010). It has been shown that there is a correlationbetween VEGF, neuronal growth factor (NGF) and ET_(B) receptors in thedeveloping brain. ET_(B) receptors can be stimulated by administeringET_(B) receptor agonists such as IRL-1620 and growth of the CNS treatdiseases can be promoted where CNS has been damaged or has not grownappropriately.

Neurodegenerative Diseases

Neurodegeneration is a term for the progressive loss of structure orfunction of neurons, including death of neurons. Many neurodegenerativediseases including Amyotrophic lateral sclerosis (ALS), Parkinson's,Alzheimer's, and Huntington's occur as a result of neurodegenerativeprocesses. As research progresses, many similarities appear that relatethese diseases to one another on a sub-cellular level. Discovering thesesimilarities offers hope for therapeutic advances that could amelioratemany diseases simultaneously. There are many parallels between differentneurodegenerative disorders including atypical protein assemblies aswell as induced cell death (Bredesen et al., 2006; Rubinsztein, 2006).

Alzheimer's disease (AD) is a neurodegenerative disorder characterizedby cerebrovascular and neuronal dysfunctions leading to a progressivedecline in cognitive functions. Neuropathological hallmarks of ADinclude beta amyloid (Aβ) plaques and neurofibrillary tangles (Johnsonet al., 2008). It has long been speculated that cerebrovasculardysfunction contributes to AD. Aβ has been shown to decrease myogenicresponse, cerebral blood flow (CBF) and vasodilator responses (Han etal., 2008; Niwa et al., 2000; Paris et al., 2004; Shin et al., 2007).Regulation of CBF tends to be impaired in transgenic mice with highintracerebral levels of Aβ (Niwa et al., 2002). Synthetic Aβ has beenshown to impair endothelin (ET) dependent relaxation and enhancevasoconstriction in vivo and in vitro (Niwa et al., 2000; Niwa et al.,2001).

Several studies have demonstrated an involvement of ET in AD. ET is anendogenous vasoregulatory peptide which targets two mainreceptors—ET_(A) and ET_(B). ET_(A) receptors are mainly located onvascular smooth muscle cells and mediate vasoconstriction, whereasET_(B) receptors are mainly located on vascular endothelial cells andmediate vasodilatation (Goto et al., 1989; Tsukahara et al., 1994). EThas been demonstrated to be present in the brain and plays an importantrole in the regulation of cerebral and systemic blood circulation(Gulati et al., 1997; Gulati et al., 1996; Gulati et al., 1995; Rebelloet al., 1995a). It was initially demonstrated that ET-1 concentrationsin the cerebrospinal fluid of patients with AD were lower compared tocontrol (Yoshizawa et al., 1992), however, subsequent studies indicatethat ET-1 like immunoreactivity was significantly increased in thecerebral cortex (frontal and occipital lobes) of patients that sufferedfrom AD compared to control brains (Minami et al., 1995). Brain samplesof AD patients obtained post mortem showed increased expression of ET-1immunoreactivity in astrocytes (Zhang et al., 1994). It has beensuggested that ET-1 released from astrocytes may reach the vascularsmooth muscle cells and induce vasoconstriction. ET binding sites in thehuman brains with AD were found to be decreased which could be due toloss of neurons in the cortex (Kohzuki et al., 1995).

The mechanism by which soluble Aβ interferes with vascular function isnot fully understood. A possible mechanism by which soluble Aβinterferes with vascular function may be mediated through ET-1 whichplays a central role in the regulation of cardiovascular functions andregional blood flow (Gulati et al., 1997; Gulati et al., 1996; Gulati etal., 1995). It was previously found that specific ET_(A) receptorantagonists (BMS182874 and BQ123) prevent Aβ induced oxidative stressand cognitive deficits (Briyal et al., 2011). Specific ET_(A) receptorantagonists reduced escape latencies and also increased preference forthe target quadrant. On the other hand, a nonspecific ET_(A)/ET_(B)receptor antagonist (TAK-044) did not produce any improvement in spatialmemory deficit or loss of preference for the target quadrant (Briyal etal., 2011). This lack of improvement with the non-specific ET_(A)/ET_(B)antagonist indicated to us the specific involvement of ET_(B) receptorsin AD.

ET binding sites in the brain are predominantly of ET_(B) receptors, andET_(B) receptor agonists have been shown to be anti-apoptotic againstneurotoxicity of Aβ (Yagami et al., 2002). Complete deficiency orblockade of ET_(B) receptors leads to exacerbation of ischemic braindamage, possibly due to the shift in ET vasomotor balance (Chuquet etal., 2002; Ehrenreich et al., 1999). It has been demonstrated thatactivation of ET_(B) receptors with intravenous IRL-1620, a highlyselective ET_(B) agonist, results in a significant elevation in CBF innormal rats and reduction in neurological deficit and infarct volume ofstroked rats (Leonard et al., 2011; Leonard and Gulati, 2009). It wasfurther found that the efficacy of IRL-1620 in a rat model of stroke wascompletely antagonized by BQ788 indicating an involvement of ET_(B)receptors (Leonard et al., 2011; 2012).

Stroke and Cerebrovascular Disorders

Stroke is the rapid loss of brain function due to disturbance in theblood supply to the brain, which can be due to ischemia or a hemorrhage(Sims and Muyderman, 2009). It is the second leading cause of death andthe fourth leading cause of disability worldwide (Mathers et al., 2009;Strong et al., 2007). It is also a predisposing factor for epilepsy,falls and depression (Fisher and Norrving, 2011) and is a foremost causeof functional impairments, with 20% of survivors requiring institutionalcare after 3 months and 15%-30% being permanently disabled (Steinwachset al., 2000).

Stroke is divided into two broad categories: Ischemic strokes, caused bysudden occlusion of arteries supplying the brain, either due to athrombus at the site of occlusion or formed in another part of thecirculation. According to recent data released by the American HeartAssociation, 87% of strokes are classified as ischemic (Deb et al.,2010; Feigin et al., 2009; Roger et al., 2012). Hemorrhagic strokes,caused by bleeding from one of the brain's arteries into the braintissue (subarachnoid hemorrhage) or arterial bleeding in the spacebetween meninges (intra-cerebral hemorrhage).

The outcome after a stroke depends on the site and severity of braininjury. A very severe stroke can cause sudden death. Stroke affectedarea of the brain cannot function, which may result in an inability tomove one or more limbs on one side of the body, inability to understandor formulate speech, or an inability to see one side of the visual field(Bath and Lees, 2000; Donnan et al., 2008). Early recognition of strokeis most important in order to expedite diagnostic tests and treatments.

Despite the severity of ischemic stroke, the only currently availableFDA-approved pharmacological treatment is recombinant tissue plasminogenactivator (rtPA), which dissolves the clot and restores blood flow tothe brain. This treatment is complicated by the relatively short windowof time between infarct and treatment (3-4 h) and the increased risk ofsubarachnoid hemorrhage (Micieli et al., 2009). A large number of otheragents, broadly classified as neuroprotective and aiming to slow or stopthe secondary damage associated with the ischemic cascade followingstroke, have shown promise in the initial stages of research but havethus far failed to demonstrate efficacy in clinical trials (Ly et al.,2006). A new approach is therefore needed, one which has the potentialto address both the restoration of blood flow and attenuate secondarydamage to the penumbral area.

Following both ischemic stroke and subarachnoid hemorrhage, ET levels inthe blood and ET immunoreactivity in the tissues are elevated (Asano etal., 1990; Rebello et al., 1995b; Viossat et al., 1993). A demonstrationthat the increase in ET levels coincides with a decrease in regionalblood flow in the ischemic areas of the brain following experimentalstroke led to the investigation of several ET antagonists in thetreatment of focal ischemic stroke (Patel et al., 1995). Although someETA specific and ETA/B non-specific antagonists have shown promise inexperimental stroke models, others have not (Barone et al., 2000; Baroneet al., 1995; Briyal and Gulati, 2010; Briyal et al., 2007; Briyal etal., 2012a; Gupta et al., 2005; Kaundal et al., 2012; Zhang et al.,2008; Zhang et al., 2005). Overall, this approach has not been useful.It has been demonstrated that ET_(B) receptors, which increase VEGF andNGF in the brain, are overexpressed at the time of birth and theirexpression decreases with maturity of the brain (Briyal et al., 2012b).It appears that ET_(B) receptors present in large number in the CNS playa key role in its development. This fundamental information demonstratesthe possible involvement of ET_(B) receptor in the brain development togenerate neurovascular plasticity of the brain that has been damagedfollowing cerebral ischemia. It was found that stimulation of ET_(B)receptors with intravenous IRL-1620, a highly selective ET_(B) agonist,resulted in a significant elevation in cerebral blood flow in normalrats (Leonard and Gulati, 2009). In addition, functional ET_(B)receptors have been shown to enhance proliferation of neuronalprogenitors and to protect against apoptosis in the dentate gyrus,olfactory epithelium, and cortical neurons (Ehrenreich et al., 1999;Laziz et al., 2011; Lee et al., 2003; Yagami et al., 2005). The evidencethat a deficiency in ET_(B) receptors leads to a poorer outcomefollowing cerebral ischemia (Chuquet et al., 2002) and completedeficiency or blockade of ET_(B) receptors leads to exacerbation ofischemic brain damage (Ehrenreich et al., 1999) led to the investigationof the role of ET_(B) receptors in a model of ischemic stroke. When amajority of research on ET and stroke thus far has focused onantagonizing ET_(A) receptors selectively or non-selectively in order toprevent excessive vasoconstriction, the effect of selectively activatingETB receptors in a focal stroke model was examined (Leonard et al.,2011; 2012; Leonard and Gulati, 2013).

In clinical practice at present there are two basic treatments,preventive treatment using long term antiplatelet or anticoagulantagents to reduce the risk of stroke, or acute treatment byfibrinolytics. However, less than 2% of patients are able to receivefibrinolytics (Font et al., 2010). Extensive research is being conductedin search of neuroprotective agents for possible use in acute phase ofstroke, and of agents that can be used for neurorepair in later stagesof stroke.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for treatingneuropsychiatric disorders in vertebrates and humans. More specifically,the present invention provides for use of IRL-1620, an endothelin-Breceptor agonist, in appropriate doses to be a neuroprotective and aneuroregenerative agent.

Accordingly, in one aspect the disclosure provides a method of treatinga neuropsychiatric disorder comprising administering to a patient inneed thereof a therapeutically effective amount of an endothelin-Breceptor agonist to treat the neuropsychiatric disorder.

In some embodiments, the endothelin-B receptor agonist isco-administered with an additional agent to treat the neuropsychiatricdisorder. In some embodiments, the additional agent is selected from thegroup consisting of an antidepressant, an anti-inflammatory agent, a CNSstimulant, a neuroleptic, and an anti-proliferative agent.

In additional embodiments, the endothelin-B receptor agonist is selectedfrom the group consisting of IRL-1620, BQ-3020,[Ala^(1,3,11,15)]-Endothelin, Sarafotoxin S6c, endothelin-3, and amixture thereof.

The present disclosure contemplates that in further embodiments, theneuropsychiatric disorder is selected from the group consisting of acerebrovascular disease, stroke, cerebral ischemia, cerebral hemorrhage,head trauma, brain injury, a brain tumor, multiple sclerosis anddemyelinating diseases, dementia, vascular dementia, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, ataxia, motor neurondisease, Amyotrophic lateral sclerosis, drug intoxication, alcoholism,chronic brain infections, brain abscess, white matter disease,Binswanger's disease, Moyamoya disease, perinatal hypoxia, cerebralasphyxia, intracranial birth injury, congenital malformation of thebrain, mood disorders, and depression.

In some embodiments, the endothelin-B receptor agonist is administeredat a dose ranging from 0.0001 to 0.5 mg/kg. In further embodiments, theendothelin-B receptor agonist is administered repeatedly at intervals of1 to 6 hours after every two to five days.

In any of the embodiments of the disclosure, it is contemplated that thepatient is a mammal. In some embodiments, the mammal is a human.

In another aspect, the disclosure provides a composition comprising (a)an endothelin-B receptor agonist, (b) an agent used to treat aneuropsychiatric disorder, and optionally (c) an excipient.

The disclosure also provides, in an additional aspect, an article ofmanufacture comprising (a) a packaged composition comprising anendothelin-B receptor agonist and an agent for a neuropsychiatricdisorder; (b) an insert providing instructions for a simultaneous orsequential administration of the endothelin-B receptor agonist and theagent for the neuropsychiatric disorder to treat a patient in needthereof; and (c) a container for (a) and (b). In some embodiments, theendothelin-B receptor agonist is IRL-1620.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structure of IRL-1620(Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp; SEQ ID NO:1).

FIGS. 2A-2D. Expression of vascular endothelial growth factor andendothelin B receptors in the vasculature of the postnatal rat brain. A)Representative images of blood vessels in the rat cortex at postnataldays 1, 7, 14 and 28, stained for VEGF (red) and ET_(B) receptors(green). Scale bar=10 μm. B) Intensity of VEGF in the rat brainvasculature at postnatal days 1, 7, 14 and 28. C) Number ofVEGF-positive vessels per 20 μm-thick rat brain slice at postnatal days1, 7, 14 and 28. D) Intensity of ET_(B) receptors in the rat brainvasculature at postnatal days 1, 7, 14 and 28. *P<0.05 vs. day 1;#P<0.01 vs. day 7. Values are expressed as mean±SEM. 30 male pups weredivided into following groups: Day 1 (N=10); Day 7 (N=10); Day 14 (N=5);Day 28 (N=5).

FIGS. 3A-3E. Expression of nerve growth factor and endothelin Breceptors in the cortex and subventricular zone of the postnatal ratbrain. A) Representative images of the rat cortex at postnatal days 1,7, 14 and 28, stained for NGF (red) and ET_(B) receptors (green). Scalebar=100 μm. B) Representative images of the rat SVZ at postnatal days 1,7, 14 and 28, stained for NGF (red) and ET_(B) receptors (green). Scalebar=100 μm. C) Intensity of NGF in the cortex and SVZ of the rat brainat postnatal days 1, 7, 14 and 28. D) Number of NGF-positive cells per100 μm² in the cortex and SVZ of the rat brain at postnatal days 1, 7,14 and 28. E) Intensity of ET_(B) receptors in the cortex and SVZ of therat brain at postnatal days 1, 7, 14 and 28. *P<0.001 vs. day 1; #P<0.01vs. day 7. Values are expressed as mean±SEM. 30 male pups were dividedinto following groups: Day 1 (N=10); Day 7 (N=10); Day 14 (N=5); Day 28(N=5).

FIGS. 4A-4D. Effect of ET_(B) receptor agonist, IRL-1620, on vascularendothelial growth factor and ET_(B) expression in the postnatal ratbrain. Rat pups were administered either saline (control; N=5) orIRL-1620 (5 μg/kg, IV; N=5) on postnatal day 21. Pups were thensacrificed on postnatal day 28 and brains removed for analysis. A)Representative images of blood vessels in the control andIRL-1620-treated rat cortex at postnatal day 28, stained for VEGF (red)and ET_(B) receptors (green). Scale bar=10 μm. B) Intensity of VEGF inthe rat brain vasculature at postnatal day 28. C) Number ofVEGF-positive vessels per 20 μm-thick rat brain slice at postnatal day28. D) Intensity of ET_(B) receptors in the rat brain vasculature atpostnatal day 28. *P<0.05 vs. Control. Values are expressed as mean±SEM.

FIGS. 5A-5E. Effect of ET_(B) receptor agonist, IRL-1620, on nervegrowth factor and ET_(B) expression in the postnatal rat brain. Rat pupswere administered either saline (control; N=5) or IRL-1620 (5 μg/kg, IV;N=5) on postnatal day 21. Pups were then sacrificed on postnatal day 28and brains removed for analysis. A) Representative images of the cortexof control and IRL-1620-treated rat brains at postnatal day 28, stainedfor NGF (red) and ET_(B) receptors (green). Scale bar=100 μm. B)Representative images of the SVZ of control and IRL-1620-treated ratbrains at postnatal day 28, stained for NGF (red) and ET_(B) receptors(green). Scale bar=100 μm. C) Intensity of NGF in the cortex and SVZ ofthe rat brain at postnatal day 28. D) Number of NGF-positive cells per100 μm² in the cortex and SVZ of the rat brain at postnatal day 28. E)Intensity of ET_(B) receptors in the cortex and SVZ of the rat brain atpostnatal day 28. *P<0.05 vs. Control. Values are expressed as mean±SEM.

FIGS. 6A-6D. Effect of ET_(B) receptor agonist, IRL-1620, on proteinlevels of vascular growth factor, nerve growth factor and ET_(B)receptors in the postnatal rat brain. Rat pups were administered eithersaline (control; N=5) or IRL-1620 (5 μg/kg, IV; N=5) on postnatal day21. Pups were then sacrificed on postnatal day 28 and brains removed foranalysis. A) Representative blots of VEGF, ET_(B) and NGF proteinexpression in the control and IRL-1620 treated rat brains at postnatalday 28 with either β-tubulin or β-actin as protein loading controls.Lane 1=Control; Lane 2=IRL-1620. B) Fold change in the expression ofVEGF in the rat brain at postnatal day 28. C) Fold change in theexpression of ET_(B) receptors in the rat brain at postnatal day 28. D)Fold change in the expression of NGF in the rat brain at postnatal day28. *P<0.05 vs. Control. Values are expressed as mean±SEM.

FIGS. 7A-7C. Effect of an ET_(B) receptor agonist, IRL-1620, in presenceand absence of an ET_(B) receptor antagonist, BQ788, on malondialdehyde(MDA) (A), reduced glutathione (GSH) (B) and superoxide dismutase (SOD)(C) levels in Aβ induced oxidative stress in the rat brain. Values areexpressed as mean±SEM. *p<0.0001 compared to sham; ^(#)p<0.001 comparedto Aβ+vehicle (N=6).

FIGS. 8A-8B. Effect of an ET_(B) receptor agonist, IRL-1620, in presenceand absence of an ET_(B) receptor antagonist, BQ788, on the escapelatency (A) and path length (B) on each training day of the water mazetask in non-diabetic rats. The animals were submitted to four dailytrials to find a hidden platform for 4 training days. Values wereexpressed as mean±S.E.M. *p<0.001 compared to sham; ^(#)p<0.001 comparedto Aβ+vehicle (N=6).

FIGS. 9A-9B. Effect of an ET_(B) receptor agonist, IRL-1620, in presenceand absence of an ET_(B) receptor antagonist, BQ788, in the water mazeprobe trial task. Time spent in each quadrant in the probe trial innon-diabetic rats (A). Representative trajectories of each group duringprobe trial in the water maze task (B). Values were expressed asmean±S.E.M. *p<0.001 compared to sham; #p<0.001 compared to Aβ+vehicle(N=6).

FIGS. 10A-10B. Effect of an ET_(B) receptor agonist, IRL-1620, inpresence and absence of an ET_(B) receptor antagonist, BQ788, on theescape latency (A) and path length (B) on each training day of the watermaze task in diabetic rats. The animals were submitted to four dailytrials to find a hidden platform for 4 training days. Values wereexpressed as mean±S.E.M. *p<0.001 compared to sham; #p<0.001 compared toAβ+vehicle (N=6).

FIGS. 11A-11B. Effect of an ET_(B) receptor agonist, IRL-1620, inpresence and absence of an ET_(B) receptor antagonist, BQ788, in thewater maze probe trial task. Time spent in each quadrant in the probetrial in diabetic rats (A). Representative trajectories of each groupduring probe trial in the water maze task (B). Values were expressed asmean±S.E.M. *p<0.001 compared to sham; ^(#)p<0.001 compared toAβ+vehicle (N=6).

FIG. 12: Two mm coronal sections of brains stained with TTC to visualizethe infarct area 7 days post middle cerebral artery occlusion (redindicates normal tissue and white indicates infarct tissue).Representative slices from groups are shown. IRL-1620 (5 mg/kg, IV) orisotonic saline (1 ml/kg, IV) was injected at 2, 4, and 6 hr post MCAO.BQ-788 (1 mg/kg, IV) was administered once 15 min prior to the firstinjection of IRL-1620 or vehicle.

FIG. 13: Effect of ET_(B) receptor agonist, IRL-1620, in presence andabsence of BQ788 on 7 day survival of rats undergoing either shamsurgery or middle cerebral artery occlusion.

FIGS. 14A-14B: Binding affinity (K_(d)) and receptor density (B_(max))of ET_(B) receptors in the left and right cerebral hemisphere in maleSprague Dawley rats (A) 24-hours and (B) 7 days following MCAO. Valuesare expressed as Mean±S.E.M, N=4 each group. No significant change inK_(d) or B_(max) was observed between left and right hemispheres in bothsham (control) and stroke (MCAO) groups.

FIG. 15: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), and antagonist, BQ788(1 mg/kg, i.v.), on glial fibrillary acidic protein (GFAP) post middlecerebral artery occlusion. A. Representative 30 μm-thick ischemic brainslice stained for the ET_(B) receptor (green) and GFAP (red). Scalebar=2000 μm. Bottom panel shows number of reactive astrocytes(GFAP+cells) per 100 μm² in the cortex, striatum, and subventricularzone of middle cerebral artery occluded rats at 24 h after infarct.*P<0.001 vs. sham. #P<0.0001 vs. MCAO+vehicle. @P<0.01 vs.MCAO+IRL-1620; and number of reactive astrocytes (GFAP+cells) per 100μm² in the cortex, striatum, and subventricular zone of middle cerebralartery occluded rats at 1 w after infarct. Values are expressed asmean±SEM (n=5/group). *P<0.001 vs. sham.

FIGS. 16A-16D: Effect of ETB receptor agonist, IRL-1620 (three doses of5 μg/kg, i.v., at 2, 4 and 6 h post ischemia), and antagonist, BQ788 (1mg/kg, i.v.), on neuronal nuclei (NeuN) post middle cerebral arteryocclusion. (A) Representative image of the cortex of an IRL-1620-treatedanimal 1 week following MCAO, stained for the ETB receptor (green) andNeuN (red). Scale bar=100 μm. (B) Representative image of the striatumof an IRL-1620-treated animal 1 week following MCAO, stained for the ETBreceptor (green) and NeuN (red). Scale bar=10 μm. (C) Number of neuronalnuclei per 100 μm² in the cortex, striatum, and subventricular zone ofmiddle cerebral artery occluded rats at 24 h after infarct. *P<0.05 vs.sham. ^(#)P<0.01 vs. MCAO+vehicle. P<0.0001 vs. MCAO+IRL-1620. (D)Number of neuronal nuclei per 100 μm² in the cortex, striatum, andsubventricular zone of middle cerebral artery occluded rats at 1 weekafter infarct. Values are expressed as mean±SEM (n=5/group). *P<0.0001vs. sham. ^(#)P<0.0001 vs. MCAO+vehicle. P<0.0001 vs. MCAO+IRL-1620.

FIG. 17: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), and antagonist, BQ788(1 mg/kg, i.v.), on proliferating cells post middle cerebral arteryocclusion. Representative image of the cortex of an IRL-1620-treatedanimal 1 week following MCAO depicting a cerebral blood vessel, stainedfor the ET_(B) receptor (green) and BrdU (red). Scale bar=100 μm. Bottompanel shows number of proliferating cells (BrdU+) per 100 μm² in thecortex, striatum, and subventricular zone of middle cerebral arteryoccluded rats at 1 week after infarct. Values are expressed as mean±SEM(n=5/group). *P<0.01 vs. sham. #P<0.0001 vs. MCAO+vehicle. @P<0.0001 vs.MCAO+IRL-1620.

FIGS. 18A-18B: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), and antagonist, BQ788(1 mg/kg, i.v.), on vascular endothelial growth factor (VEGF) postmiddle cerebral artery occlusion. A. Representative images of bloodvessels in the rat cortex 24 h following MCAO, stained for the ET_(B)receptor (green) and VEGF (red). Rows: 1. Sham; 2. MCAO+vehicle; 3.MCAO+IRL-1620; 4. MCAO+BQ788+vehicle; 5. MCAO+BQ788+IRL-1620. Scalebar=10 μm. B. Representative images of blood vessels in the rat cortex 1week following MCAO, stained for the ET_(B) receptor (green) and VEGF(red). Rows same as in (A 24 h). Scale bar=10 μm.

FIG. 19: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), and antagonist, BQ788(1 mg/kg, i.v.), on vascular endothelial growth factor (VEGF) postmiddle cerebral artery occlusion. Upper panel shows number of VEGF+vessels per 30 μm brain slice middle cerebral artery occluded rats at 24h after infarct. *P<0.05 vs. sham. #P<0.01 vs. MCAO+vehicle. @P<0.05 vs.MCAO+IRL-1620. The lower panel shows number of VEGF+ vessels per 30 μmbrain slice middle cerebral artery occluded rats at 1 week afterinfarct. Values are expressed as mean±SEM (n=5/group). *P<0.01 vs. sham.#P<0.01 vs. MCAO+vehicle. @P<0.05 vs. MCAO+IRL-1620.

FIGS. 20A-20D: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), on protein levels ofvascular endothelial growth factor (VEGF) post middle cerebral arteryocclusion. A. Representative blot of VEGF protein levels in the ratbrain 24 hours post MCAO with β-tubulin as a loading control. Lane1—sham (LH), Lane 2—sham (RH), Lane 3—MCAO+Vehicle (LH), Lane4—MCAO+Vehicle (RH), Lane 5—MCAO+IRL-1620 (LH), Lane 6—MCAO+IRL-1620(RH). LH=left hemisphere, RH=right hemisphere. B. Representative blot ofVEGF protein levels in the rat brain 1 week post MCAO with β-tubulin asa loading control, with lane distribution the same as in (A). C.Expression of VEGF protein levels in the rat brain 24 hours followingMCAO. D. Expression of VEGF protein levels in the rat brain 1 weekfollowing MCAO. Values are expressed as mean±SEM (n=5/group). *P<0.001vs. Sham. #P<0.01 vs MCAO+Vehicle.

FIGS. 21A-21B: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5mg/kg, i.v., at 2, 4 and 6 hours post ischemia), and antagonist, BQ788(1 mg/kg, i.v.), on nerve growth factor (NGF) post middle cerebralartery occlusion. A. Representative image of the cortex of anIRL-1620-treated animal 1 week following MCAO, stained for the ET_(B)receptor (green) and NGF (red). Scale bar=10 μm. B. Number of NGF+ cellsper 100 μm² in the cortex, striatum, and subventricular zone of middlecerebral artery occluded rats at 1 week after infarct. Values areexpressed as mean±SEM (n=5/group). #P<0.0001 vs. MCAO+vehicle. @P<0.0001vs. MCAO+IRL-1620.

FIGS. 22A-22D: Effect of ET_(B) receptor agonist, IRL-1620 (3 doses of 5μg/kg, i.v., at 2, 4 and 6 hours post ischemia), on protein levels ofnerve growth factor (NGF) post middle cerebral artery occlusion. A.Representative blot of NGF protein levels in the rat brain 24 hours postMCAO with β-tubulin as a loading control. Lane 1—sham (LH), Lane 2—sham(RH), Lane 3—MCAO+Vehicle (LH), Lane 4—MCAO+Vehicle (RH), Lane5—MCAO+IRL-1620 (LH), Lane 6—MCAO+IRL-1620 (RH). LH=left hemisphere,RH=right hemisphere. B. Representative blot of NGF protein levels in therat brain 1 week post MCAO with β-tubulin as a loading control, withlane distribution the same as in (A). C. Expression of NGF proteinlevels in the rat brain 24 hours following MCAO. D. Expression of NGFprotein levels in the rat brain 1 week following MCAO. Values areexpressed as mean±SEM (n=5/group). *P<0.01 vs. Sham.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions and methods for treating aneuropsychiatric disorder. It is disclosed herein that an endothelin-Breceptor agonist functions as neuroprotective and a neuroregenerativeagent. The compositions and methods of the disclosure generally relateto administering an endothelin-B receptor agonist to a patient in needthereof to treat a neuropsychiatric disorder.

Accordingly, in one aspect the disclosure provides a method of treatinga neuropsychiatric disorder comprising administering to a patient inneed thereof a therapeutically effective amount of an endothelin-Breceptor agonist to treat the neuropsychiatric disorder.

In some embodiments, the endothelin-B receptor agonist is selected fromthe group consisting of IRL-1620, BQ-3020, [Ala^(1,3,11,15)]-Endothelin,Sarafotoxin S6c, endothelin-3, and a mixture thereof.

In some embodiments, the present disclosure contemplates that ET_(B)receptor agonists such as IRL-1620 (FIG. 1) can be used to treat variousneuropsychiatric disorders such as cerebrovascular diseases, stroke,cerebral ischemia, cerebral hemorrhage, head trauma, brain injury, braintumors, multiple sclerosis and demyelinating diseases, dementia,vascular dementia, Alzheimer's disease, Parkinson's disease,Huntington's disease, ataxias, motor neuron disease, Amyotrophic lateralsclerosis, drug intoxication, alcoholism, chronic brain infections,brain abscess, white matter disease, Binswanger's disease, Moyamoyadisease, perinatal hypoxia, cerebral asphyxia, intracranial birthinjury, congenital malformation of the brain, mood disorders, anddepression.

In some embodiments, the endothelin-B receptor agonist isco-administered with an additional agent to treat the neuropsychiatricdisorder. In some embodiments, the additional agent is selected from thegroup consisting of an antidepressant, an anti-inflammatory agent, a CNSstimulant, a neuroleptic, and an anti-proliferative agent.

General Additional Agents

Examples of antidepressants, CNS stimulants and neuroleptic agentsuseful in the methods and compositions of the disclosure include, butare not limited to Abilify, Adapin, Adderall, Alepam, Alertec,Aloperidin, Alplax, Alprax, Alprazolam, Alviz, Alzolam, Amantadine,Ambien, Amisulpride, Amitriptyline, Amoxapine, Amfebutamone, Anafranil,Anatensol, Ansial, Ansiced, Antabus, Antabuse, Antideprin, Anxiron,Apo-Alpraz, Apo-Primidone, Apo-Sertral, Aponal, Apozepam, Aripiprazole,Aropax, Artane, Asendin, Asendis, Asentra, Ativan, Atomoxetine, Aurorix,Aventyl, Axoren, Baclofen, Beneficat, Benperidol, Bimaran, Bioperidolo,Biston, Brotopon, Bespar, Bupropion, Buspar, Buspimen, Buspinol,Buspirone, Buspisal, Cabaser, Cabergoline, Calepsin, Calcium carbonate,Calcium carbimide, Calmax, Carbamazepine, Carbatrol, Carbolith, Celexa,Chloraldurat, Chloralhydrat, Chlordiazepoxide, Chlorpromazine,Cibalith-S, Cipralex, Citalopram, Clomipramine, Clonazepam, Clozapine,Clozaril, Concerta, Constan, Convulex, Cylert, Cymbalta, Dapotum,Daquiran, Daytrana, Defanyl, Dalmane, Damixane, Demolox, Depad,Depakene, Depakote, Depixol, Desyrel, Dostinex, dextroamphetamine,Dexedrine, Diazepam, Didrex, Divalproex, Dogmatyl, Dolophine,Droperidol, Desoxyn, Edronax, Effectin, Effexor (Efexor), Eglonyl,Einalon S, Elavil, Elontril, Endep, Epanutin, Epitol, Equetro,Escitalopram, Eskalith, Eskazinyl, Eskazine, Etrafon, Eukystol,Eunerpan, Faverin, Fazaclo, Fevarin, Finlepsin, Fludecate, Flunanthate,Fluoxetine, Fluphenazine, Flurazepam, Fluspirilene, Fluvoxamine,Focalin, Gabapentin, Geodon, Gladem, Glianimon, Guanfacine, Halcion,Halomonth, Haldol, Haloperidol, Halosten, Imap, Imipramine, Imovane,Janimine, Jatroneural, Kalma, Keselan, Klonopin, Lamotrigine, Largactil,Levomepromazine, Levoprome, Leponex, Lexapro, Libotryp Libritabs,Librium, Linton, Liskantin, Lithane, Lithium, Lithizine, Lithobid,Lithonate, Lithotabs, Lorazepam, Loxapac, Loxapine, Loxitane, Ludiomil,Lunesta, Lustral, Luvox, Lyrica, Lyogen, Manegan, Manerix, Maprotiline,Mellaril, Melleretten, Melleril, Melneurin, Melperone, Meresa,Mesoridazine, Metadate, Methamphetamine, Methotrimeprazine, Methylin,Methylphenidate, Minitran, Mirapex, Mirapexine, Moclobemide, Modafinil,Modalina, Modecate, Moditen, Molipaxin, Moxadil, Murelax, Myidone,Mylepsinum, Mysoline, Nardil, Narol, Navane, Nefazodone, Neoperidol,Neurontin, Nipolept, Norebox, Normison, Norpramine, Nortriptyline,Novodorm, Nitrazepam, Olanzapine, Omca, Oprymea, Orap, Oxazepam,Pamelor, Parnate, Paroxetine, Paxil, Peluces, Pemoline, Pergolide,Permax, Permitil, Perphenazine, Pertofrane, Phenelzine, Phenytoin,Pimozide, Piportil, Pipotiazine, Pragmarel, Pramipexole, Pregabalin,Primidone, Prolift, Prolixin, Promethazine, Prothipendyl, Protriptyline,Provigil, Prozac, Prysoline, Psymion, Quetiapine, Ralozam, Reboxetine,Redeptin, Resimatil, Restoril, Restyl, Rhotrimine, riluzole, Risperdal,Risperidone, Rispolept, Ritalin, Rivotril, Rubifen, Rozerem, Sediten,Seduxen, Selecten, Serax, Serenace, Serepax, Serenase, Serentil,Seresta, Serlain, Serlift, Seroquel, Seroxat, Sertan, Sertraline,Serzone, Sevinol, Sideril, Sifrol, Sigaperidol, Sinequan, Sinqualone,Sinquan, Sirtal, Solanax, Solian, Solvex, Songar, Stazepin, Stelazine,Stilnox, Stimuloton, Strattera, Sulpiride, Sulpiride Ratiopharm,Sulpiride Neurazpharm, Surmontil, Symbyax, Symmetrel, Tafil, Tavor,Taxagon, Tegretol, Telesmin, Temazepam, Temesta, Temposil, Terfluzine,Thioridazine, Thiothixene, Thombran, Thorazine, Timonil, tissueplasminogen activator (tPA), Tofranil, Tradon, Tramadol, Tramal,Trancin, Tranax, Trankimazin, Tranquinal, Tranylcypromine, Trazalon,Trazodone, Trazonil, Trialodine, Trevilor, Triazolam, Trifluoperazine,Trihexane, Trihexyphenidyl, Trilafon, Trimipramine, Triptil, Trittico,Troxal, Tryptanol, Tryptomer, Ultram, Valium, Valproate, Valproic acid,Valrelease, Vasiprax, Venlafaxine, Vestra, Vigicer, Vivactil,Wellbutrin, Xanax, Xanor, Xydep, Zaleplon, Zamhexal, Zeldox, Zimovane,Zispin, Ziprasidone, Zolarem, Zoldac, Zoloft, Zolpidem, Zonalon,Zopiclone, Zotepine, Zydis, and Zyprexa.

Anti-Inflammatory Agents

Any agents having anti-inflammatory effects can be used in the presentinvention. The anti-inflammatory agent can be a steroidalanti-inflammatory agent, a nonsteroidal anti-inflammatory agent, or acombination thereof. In some embodiments, anti-inflammatory agentsinclude, but are not limited to, alclofenac, alclometasone dipropionate,algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenacsodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen,apazone, balsalazide disodium, bendazac, benoxaprofen, benzydaminehydrochloride, bromelains, broperamole, budesonide, carprofen,cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasonebutyrate, clopirac, cloticasone propionate, cormethasone acetate,cortodoxone, deflazacort, desonide, desoximetasone, dexamethasonedipropionate, diclofenac potassium, diclofenac sodium, diflorasonediacetate, diflumidone sodium, diflunisal, difluprednate, diftalone,dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium,epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen,fenclofenac, fenclorac, fendo sal, fenpipalone, fentiazac, flazalone,fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin,flunixin meglumine, fluocortin butyl, fluorometholone acetate,fluquazone, flurbiprofen, fluretofen, fluticasone propionate,furaprofen, furobufen, halcinonide, halobetasol propionate, halopredoneacetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol,ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole,intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen,lofemizole hydrochloride, lomoxicam, loteprednol etabonate,meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate,mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate,morniflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone,olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone,paranyline hydrochloride, pentosan polysulfate sodium, phenbutazonesodium glycerate, pirfenidone, piroxicam, piroxicam cinnamate, piroxicamolamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone,proxazole, proxazole citrate, rimexolone, romazarit, salcolex,salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin,sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate,tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide,tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium,triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin(acetylsalicylic acid), salicylic acid, corticosteroids,glucocorticoids, tacrolimus, pimecorlimus, prodrugs thereof, co-drugsthereof, and combinations thereof.

Anti-Proliferative Agents

Any agents having anti-proliferative effects can be used in the presentinvention. The anti-proliferative agent can be a natural proteineousagent such as a cytotoxin or a synthetic molecule. In some embodiments,the active agents include antiproliferative substances such asactinomycin D, or derivatives and analogs thereof (synonyms ofactinomycin D include dactinomycin, actinomycin IV, actinomycin I₁,actinomycin X₁, and actinomycin CO, all taxoids such as taxols,docetaxel, and paclitaxel, paclitaxel derivatives, all olimus drugs suchas macrolide antibiotics, rapamycin, everolimus, structural derivativesand functional analogues of rapamycin, structural derivatives andfunctional analogues of everolimus, FKBP-12 mediated mTOR inhibitors,biolimus, prodrugs thereof, co-drugs thereof, and combinations thereof).Representative rapamycin derivatives include40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, or 40-O-tetrazole-rapamycin,40-epi-(N-1-tetrazolyl)-rapamycin (ABT-578 manufactured by AbbotLaboratories, Abbot Park, Ill.), prodrugs thereof, co-drugs thereof, andcombinations thereof.

The present disclosure contemplates that in further embodiments, theneuropsychiatric disorder is selected from the group consisting of acerebrovascular disease, stroke, cerebral ischemia, cerebral hemorrhage,head trauma, brain injury, a brain tumor, multiple sclerosis anddemyelinating diseases, dementia, vascular dementia, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, ataxia, motor neurondisease, Amyotrophic lateral sclerosis, drug intoxication, alcoholism,chronic brain infections, brain abscess, white matter disease,Binswanger's disease, Moyamoya disease, perinatal hypoxia, cerebralasphyxia, intracranial birth injury, congenital malformation of thebrain, mood disorders, and depression.

According to the disclosure, the endothelin-B receptor agonist may beadministered at a dose ranging from 0.0001 to 0.5 mg/kg. In furtherembodiments, the endothelin-B receptor agonist is administered at a doseranging from about 0.0001 to about 0.5 mg/kg, or from about 0.0001 toabout 0.4 mg/kg, or from about 0.0001 to about 0.3 mg/kg, or from about0.0001 to about 0.2 mg/kg, or from about 0.0001 to about 0.1 mg/kg, orfrom about 0.001 to about 0.5 mg/kg, or from about 0.001 to about 0.4mg/kg, or from about 0.001 to about 0.3 mg/kg, or from about 0.001 toabout 0.2 mg/kg, or from about 0.001 to about 0.1 mg/kg, or from about0.01 to about 0.5 mg/kg, or from about 0.01 to about 0.4 mg/kg, or fromabout 0.01 to about 0.3 mg/kg, or from about 0.01 to about 0.2 mg/kg, orfrom about 0.01 to about 0.1 mg/kg, or from about 0.0005 to about 0.5mg/kg, or from about 0.0005 to about 0.4 mg/kg, or from about 0.0005 toabout 0.3 mg/kg, or from about 0.0005 to about 0.2 mg/kg, or from about0.0005 to about 0.1 mg/kg. In additional embodiments, the endothelin-Breceptor agonist is administered at a dose of at least about 0.0001mg/kg, or at least about 0.0002 mg/kg, or at least about 0.0005 mg/kg,or at least about 0.001 mg/kg, or at least about 0.002 mg/kg, or atleast about 0.005 mg/kg, or at least about 0.007 mg/kg, or at leastabout 0.01 mg/kg, or at least about 0.02 mg/kg, or at least about 0.03mg/kg, or at least about 0.04 mg/kg, or at least about 0.05 mg/kg, or atleast about 0.06 mg/kg, or at least about 0.07 mg/kg, or at least about0.08 mg/kg, or at least about 0.09 mg/kg, or at least about 0.1 mg/kg,or at least about 0.2 mg/kg, or at least about 0.3 mg/kg, or at leastabout 0.4 mg/kg. In still further embodiments, the endothelin-B receptoragonist is administered at a dose of less than about 0.0001 mg/kg, orless than about 0.0002 mg/kg, or less than about 0.0005 mg/kg, or lessthan about 0.001 mg/kg, or less than about 0.002 mg/kg, or less thanabout 0.005 mg/kg, or less than about 0.007 mg/kg, or less than about0.01 mg/kg, or less than about 0.02 mg/kg, or less than about 0.03mg/kg, or less than about 0.04 mg/kg, or less than about 0.05 mg/kg, orless than about 0.06 mg/kg, or less than about 0.07 mg/kg, or less thanabout 0.08 mg/kg, or less than about 0.09 mg/kg, or less than about 0.1mg/kg, or less than about 0.2 mg/kg, or less than about 0.3 mg/kg, orless than about 0.4 mg/kg, or less than about 0.5 mg/kg.

The endothelin-B receptor agonist, in various embodiments, isadministered to a patient repeatedly at intervals of 1 to 6 hours. Insome embodiments, the endothelin-B receptor agonist is administered tothe patient every 1 to 5 hours, or every lto 4 hours, or every 1 to 2hours, or every hour, or every 2 hours, or every 3 hours, or every 4hours, or every 5 hours, or every 6 hours. In further embodiments, theendothelin-B receptor agonist is administered to the patient every twoto five days, or every three to five days, or every two days, or everythree days, or every four days, or every five days.

Endothelin B Receptor Ontogeny in the Postnatal Rat Brain

A decrease in expression of ET_(B) receptors in the brain of rat pups atpostnatal day 28 as measured using immunoblotting technique has beenreported (Briyal et al., 2012b). In order to determine the location ofthese receptors and their potential correlation with vascular and neuralgrowth factors in the developing brain, the brains wereimmunofluorescently labeled of rat pups at postnatal days 1, 7, 14 and28 with antibodies for ET_(B) receptors, VEGF and NGF. The intensity ofET_(B) receptor staining within the vasculature was significantly higheron day 14 compared to day 1 and day 7 of postnatal age (FIG. 2). Incontrast, intensity of ET_(B) staining in the cortex and subventricularzones of developing rat brain decreased significantly at day 14 ofpostnatal age compared to day 1 and day 7 (P<0.0001; FIG. 3). ET_(B)intensity was found to be similar at postnatal age of 14 and 28 days.These results indicate that there is indeed a decrease in the expressionof ET_(B) receptors within the neural tissue of the developing ratbrain, but not in the neurovasculature.

Vascular Endothelial Growth Factor Ontogeny in the Postnatal Rat Brain

VEGF in the vasculature of the postnatal rat brain was evaluated at day1, 7, 14 and 28 of postnatal age via immunofluorescent labeling. Asillustrated in FIG. 2, the intensity of VEGF staining in theneurovasculature steadily increased from postnatal days 1 through 14.Similarly, the number of VEGF-positive vessels per 20 μm-thick brainslice significantly (P<0.0001) increased from 2.22±0.36 on day 1 to5.69±0.74 on day 14 of postnatal age (FIG. 2). While both VEGF andET_(B) intensity and expression within the cerebral vasculature of thedeveloping rat brain increased with age, there was no significantcorrelation between the two (r²=0.8279; P=0.0901).

Nerve Growth Factor Ontogeny in the Postnatal Rat Brain

Immunofluorescent labeling was used to determine the expression anddistribution of NGF in the postnatal rat brain at days 1, 7, 14 and 28.The intensity of staining for NGF in the cortex of the developing ratbrain significantly decreased from day 1 to day 7 (P<0.001) and againfrom day 7 to day 14 (P<0.01) of postnatal age. There was no significantdifference in NGF intensity from postnatal day 14 to day 28 (FIG. 3).NGF intensity within the subventricular zone of the postnatal rat braindecreased between days 7 and 14 (P<0.01), with no further declinebetween days 14 and 28 (FIG. 3) of postnatal age. Interestingly, averagenumber of cells staining positive for NGF did not significantly alterduring the course of experiment in either the cortex or SVZ. There was,however, a significant correlation between a decrease in ET_(B)receptors and NGF in the cortex of the developing rat brain with age(r²=0.9742; P=0.0130). These results indicate that, as the rat brainmatures the overall expression of NGF declines. This decrease may becorrelated with the drop in ET_(B) receptors within the neuronal tissue.

Effect of IRL-1620 on Endothelin B Receptors in the Postnatal Rat Brain

Administration of ET_(B) receptor agonist, IRL-1620, on postnatal day 21resulted in a significant increase in ET_(B) receptors in the 28-day oldrat brain as compared to the control group. Overall, protein expressionof ET_(B) receptors, as measured using immunoblotting technique,increased in the animals who had received IRL-1620 (FIG. 6). Uponimmunofluorescent labeling of the brain slices, it was found thatintensity of ET_(B) receptor staining was significantly higher in boththe cerebral vasculature (FIG. 2) and the cortex (FIG. 3) ofIRL-1620-treated animals as compared to control (P<0.05). These resultssuggest that selective stimulation of ET_(B) receptors during neonataldevelopment may result in an upregulation of these receptors.

Effect of IRL-1620 on Vascular Endothelial Growth Factor in thePostnatal Rat Brain

As seen in FIG. 2, VEGF increases in the cerebral vasculature throughoutdevelopment in rats. Selective stimulation of ET_(B) receptors usingagonist IRL-1620 leads to a further increase in both the intensity ofVEGF staining and the number of VEGF-positive vessels (P<0.05; FIG. 4)within the postnatal rat brain when compared to control animals of thesame age. These results were confirmed using immunoblotting techniqueshowing that overall VEGF expression is significantly enhanced withinthe postnatal brains of rats treated with IRL-1620 (P<0.05; FIG. 6).These findings suggest that selective stimulation of ET_(B) receptorsduring development enhances cerebrovascular angiogenesis.

Effect of IRL-1620 on Nerve Growth Factor in the Postnatal Rat Brain

Immunofluorescent labeling of NGF significantly diminishes by postnatalday 14 in the rat brain. Administration of ET_(B) receptor agonist,IRL-1620, on postnatal day 21 did not alter either intensity of NGFstaining nor number of NGF-positive cells within the cortex orsubventricular zones of rats as compared to control (FIG. 5). Similarly,overall expression of NGF within the postnatal rat brain as measured viaimmunoblotting technique was comparable in both control and treatedgroups (FIG. 6). Selective ET_(B) receptor stimulation does not appearto have any significant effect on NGF levels within the rat brain duringpostnatal development.

Experimental Procedure Animals

Timed pregnant female Sprague-Dawley rats (Harlan, Indianapolis, Ind.)were caged singly in a room controlled for ambient temperature (23±1°C.), humidity (50±10%) and a 12 h light/dark cycle (6:00 am-6:00 pm).Food and water were available ad libitum. All animal care and use forprocedures were approved by the Institutional Animal Care and UseCommittee (IACUC) of Midwestern University. In order to avoid variationdue to hormonal changes, male pups only were separated and utilized forthis study. The pups were euthanized via decapitation on postnatal day1, 7, 14 and 28. The brains were aseptically removed and processed foreither immunofluorescent labeling or immunoblot analysis of ET_(B)receptors, VEGF and NGF.

Study Design and Agent Administration

After 21 days of gestation, 70 male and 65 female pups were born to 10pregnant female rats. 30 male pups were randomly selected for Study I,and divided into 4 groups as follows: Group 1=male pups euthanized onpostnatal day 1 (N=10); Group 2=male pups euthanized on postnatal day 7(N=10); Group 3=male pups euthanized on postnatal day 14 (N=5); Group4=male pups euthanized on postnatal day 28 (N=5). An additional 20 malepups were randomly selected for Study II, and divided into 2 groups:Control (N=10) and IRL-1620-treated (N=10). Pups in Study II received 3doses of either isotonic saline (1 ml/kg) or ET_(B) receptor agonistIRL-1620 (5 μg/kg; American Peptide Co, Inc., Sunnyvale, Calif.) onpostnatal day 21. The doses were given intravenously at 2 hourintervals. All pups were weighed and evaluated for developmental andbehavioral characteristics at postnatal day 1, 7, 14 and 28.Developmental and behavioral characteristics included active vs.sluggish behavior, healthy vs. shedding fur, licking, grooming,aggressive behavior, defecation, urination and wet dog shakes.

Immunofluorescent Labeling

Immunofluorescently labeled antibodies were used to determine theexpression of ET_(B) receptors, VEGF and NGF in the developing ratbrain. Male rat pups were euthanized via decapitation, and the brainswere removed at postnatal day 1, 7, 14 and 28. The brains were washed inisotonic saline and transferred to a 4% paraformaldehyde (PFA)/NaPO4buffer solution for 2 hours to fix the tissue, followed by suspension ina 20% sucrose/4% PFA solution for 48 hours at 4° C. Brains were thensliced into 20 μm thick coronal sections at −30° C. using a cryostat(Microtome cryostat HM 505 E; Walldorf, Germany). Sections wereprocessed for immunofluorescent staining as described by Loo, et al.(Loo et al., 2002), with minor modifications. The primary antibody forET_(B) receptors was an anti-ET_(B) receptor antibody raised in sheepagainst the carboxy-terminal peptide of the rat ET_(B) receptor (1:200;ab50658; Abcam, Cambridge, Mass.). Determination of angiogenic andneurogenic markers was performed using antibodies against VEGF(anti-VEGF; 1:500; ab46154; Abcam) and nerve growth factor (anti-NGF;ab6199; Abcam). Sections were washed in PBS and then blocked with 10%v/v serum in PBS containing 0.3% Triton X-100 for 1 h. Sections werethen incubated with the primary antibody overnight at 4° C., and againwashed with PBS and incubated with the appropriate secondary antibodyfor 2 h at room temperature. Double labeling for co-localization wasperformed sequentially. Sections were rinsed with PBS and mounted onglass slides with Vectashield (Vector Laboratories, Inc., Burlingame,Calif.). Fluorescence was detected using an inverted fluorescentmicroscope (Nikon Eclipse TiE, Melville, N.Y.). All images for analysiswere taken with the same exposure (300 msec for VEGF and NGF; 800 msecfor ET_(B)) and objective (Plan Fluor 10× Ph1DL) settings with amultichannel ND acquisition using NIS Elements BR imaging software(Nikon Instruments, Inc., Melville, N.Y.).

Immunofluorescent Analysis

Analyses for NGF were performed specifically in the cortex and SVZ ofthe rat brain. Overlaying a grid of 100×100 μm squares on each image,the number of cells staining positively for NGF was determined in sixrandomly selected, non-congruent 100 μm² sections per brain slice ineach area. All cells falling at least 50% inside the 100 μm² area werecounted. For the evaluation of angiogenesis, the total number ofVEGF-positive blood vessels was determined per brain slice. Fluorescentintensity for each marker was measured using the unaltered images withNIS Elements BR imaging software (Nikon Instruments, Inc., Melville,N.Y.).

Immunoblotting

Protein levels of VEGF, NGF and ET_(B) receptors in the postnatal ratbrain of animals in Study II were estimated using immunoblottingtechnique. Animals were decapitated and the brains were removed onpostnatal day 28, which is 7 days post administration of either salineor ET_(B) receptor agonist, IRL-1620. The tissue was homogenized in 10×(w/v) RIPA lysis buffer, and protein concentration was determinedaccording to the Lowry method, using bovine serum albumin as standard(Lowry et al., 1951). Protein (60 μg) was denatured in Laemmli samplebuffer and resolved in 10% SDS-PAGE, and then transferred ontonitrocellulose membrane. After blocking, the membranes were incubatedwith rabbit polyclonal anti-VEGF (1:1000; Abcam, Cambridge, Mass.),anti-NGF (1:500; Abcam, Cambridge, Mass.), or anti-ET_(B) (1:1000;Sigma-Aldrich) antibodies overnight at 4° C., followed by 1.5 hoursincubation with by HRP-conjugated secondary antibodies (1:2000; CellSignaling Technology, Inc., MA) at room temperature. β-tubulin (1:2000;Cell Signaling Technology, Inc. MA) or β-actin (1:10000; Sigma-Aldrich)were used as loading controls. The labeled proteins were visualized withSuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific)using the Kodak Gel Logic 1500 Imaging System (Carestream Health Inc.,New Haven, Conn.). Protein expression was analyzed using Image J (NIH)software.

Statistical Analysis

A power analysis was conducted using GraphPad Instat-2.00. The power wasset to 80% (beta=0.8) and the level of significance (alpha) used was0.05. The power analysis indicated that a sample size of 5 per group wassufficient to achieve the desired power. Data are presented asmean±S.E.M. One-way analysis of variance (ANOVA) followed by Tukey'spost-hoc comparison test was used for intergroup comparison. Unpairedt-test was used for comparison of postnatal day 28 control vs. IRL-1620groups. A P value of less than 0.05 was considered to be significant.The statistical analysis was processed using GraphPad Prism 6.02(GraphPad, San Diego, Calif.).

The goal of the present study was to determine the ontogeny of ET_(B)receptors, VEGF and NGF in the postnatal rat brain, as well as toinvestigate whether or not selective stimulation of ET_(B) receptorsduring postnatal development would result in changes in the expressionof either the receptors or growth factors. In confirmation of previousstudies (Briyal et al., 2012b), it was found that ET_(B) receptorsappear to decrease in the neuronal tissue with postnatal age.Interestingly, both ET_(B) receptor and VEGF intensity increased withinthe cerebral vasculature from postnatal day 1 to day 14. NGF, on theother hand, decreased concurrently with ET_(B) receptors in the cortexand SVZ of the rat brain from postnatal day 1 to day 14. Selectivestimulation of ET_(B) receptors via intravenous IRL-1620 on postnatalday 21 led to a significant increase in ET_(B) receptors and VEGF, butnot NGF. These results suggest that, while the ontogeny of ET_(B)receptors may be related to both vascular and neuronal growth factorswithin the developing brain, stimulation of ET_(B) receptors duringpostnatal development exerts more influence over angiogenesis thanneurogenesis.

While the rat model is commonly used in ontological studies, it isimportant to recognize that gestation and development, particularly withregards to the brain, differ between rodents and humans. It has beenreported that 16.7 rat days are equivalent to 1 human year (Quinn,2005). Extrapolating this information out onto the timeline of thepresent study provides the following information: postnatal day 1=21human days; postnatal day 7=5 human months; postnatal day 14=10 humanmonths; and postnatal day 28=20 human months. This is significant asrapid brain growth for the human begins at the end of the secondtrimester, peaks at birth and then decreases over the next severalyears. Rats, however, with a gestational period of only 21 days,experience the most rapid rise in brain growth and development withinthe first 10 postnatal days (Gil-Mohapel et al., 2010). This period isroughly equivalent to the third trimester in human brain development.

The period of time evaluated by the present study, postnatal rat days 1to 28, coincide with the first 2 years of equivalent human braindevelopment. As expected, the highest levels of neuronal growth factoralong with ET_(B) receptors were noted in the cortex and SVZ on thefirst day following birth. These levels decreased significantly withincreasing brain maturity from day 1 to day 14. No significant changewas noted between days 14 and 28. A decrease in ET_(B) receptor proteinexpression with the whole rat brain by postnatal day 21 has beenreported (Briyal et al., 2012b). The present data shed light on earlierreports of low levels of neuronal progenitors and increased CNSdisturbances in ET_(B) deficient rat pups (Ehrenreich et al., 2000;Riechers et al., 2004; Vidovic et al., 2008). A significant co-relationwas found between the declines in intensity of NGF and ET_(B) receptorstaining in the cerebral cortex of developing brain; however, whenIRL-1620 was administered on postnatal day 21, to stimulate ET_(B)receptors, this co-relation was lost and an increase in ET_(B) receptorstaining was not accompanied with an increase in NGF staining in thecerebral cortex. It is possible that there is a regulatory mechanismthat initiates a decrease in ET_(B) receptor and NGF staining as thebrain matures and this regulatory mechanism does not allow IRL-1620 toproduce any pharmacologically induced increase in ET_(B) receptor andNGF staining of the brain that is close to maturity.

ET_(B) receptor stimulation, therefore, does not appear to increaseneurogenesis during late-stage CNS development, although it has beenshown to enhance this process during adult neurovascular repairprocesses (Leonard and Gulati, 2013) indicating a loss of regulatorymechanism following cerebral ischemia. Selective stimulation of ET_(B)receptors did, however increase the overall expression of ET_(B) withinthe whole brain as measured via immunoblotting technique as well as theintensity of ET_(B) receptor staining of the cerebral vasculature of thedeveloping rat brain, suggesting that such stimulation does enhanceangiogenesis during the late postnatal period.

Overall, both VEGF and ET_(B) intensity within the cerebral vasculatureincreased throughout the period studied. VEGF is a potent angiogenicfactor essential for CNS vascularization, development and repair.Previous research has indicated the VEGF is restricted mainly tocortical neurons early in development, but then switches to maturingglial cells around the blood vessels as the vascular bed begins tostabilize (Ogunshola et al., 2000). While VEGF expression within theneuronal tissue was not specifically determined, the results demonstratean increase in VEGF around the vasculature as the brain develops. In theearlier stages of CNS development, when NGF levels are high, VEGF mayserve as a promoter of neurogenesis, neuronal migration andneuroprotection (Rosenstein et al., 2010). Similar effects are seen inthe adult brain following ischemic injury, with increased levels of VEGFpromoting cerebrovascular repair (Gora-Kupilas and Josko, 2005; Nowackaand Obuchowicz, 2012). Indeed, previous studies have shown thatselective stimulation of ET_(B) receptors following permanent cerebralischemia leads to an increase in both VEGF and ET_(B) expression,coincident with neuroprotection and cerebrovascular repair (Leonard andGulati, 2013). In the present study, selective stimulation of ET_(B)receptors at postnatal day 21 resulted in increases in VEGF and ET_(B)expression both in the cerebral vasculature and the whole brain. Theseresults serve to both confirm the relationship between ET_(B) receptorsand VEGF and to highlight their importance in the developing brain.

Hypoxia-ischemia brain damage during the neonatal period is one of themain factors in brain dysfunction (Li et al., 2008). Premature infants,in particular, often experience episodes of hypoxia-ischemia which canlead to reduced cortical growth and development. These impairments maycontinue through childhood and adolescence, and can cause dysfunctionwithin the neural micro circuitry leading to epilepsy,neurodevelopmental disorders and psychiatric illnesses (Malik et al.,2013). Episodes of hypoxia-ischemia within the brain are known toincrease hypoxia-inducible transcription factor-1, which in turnupregulates VEGF (Trollmann et al., 2008). It has been shown thatselective ET_(B) receptor stimulation upregulates both ET_(B) receptorsand VEGF in the brains of both normal neonates and adult rats subjectedto cerebral ischemia. It is possible that early treatment with aselective ET_(B) receptor agonist may enhance VEGF and neuroprotection,thereby enabling the brains of infants suffering from hypoxic damage torepair this neuronal damage.

The present study indicates that selective stimulation of ET_(B)receptors enhances VEGF within the developing rat brain. While nosimilar significant upregulation of NGF was noted in the present study,a correlation in the ontogeny of ET_(B) receptors and NGF was observedwithin the cortex and SVZ of postnatal rat pups. It appears that bothET_(B) receptors and NGF are important in the early phase of developmentof the CNS. As studies have shown that selective ET_(B) receptorstimulation is capable of enhancing cerebrovascular repair in the adultbrain following ischemia, it would be of interest to determine whetheror not similar treatment could significantly improve repair mechanismswithin the developing brain subjected to ischemia.

Studies in Animal Model of Alzheimer's Disease

Male Sprague-Dawley rats (Harlan, Indianapolis, Ind.) weighing 280-310 gwere allowed to acclimate for at least 4 days before use. Animals werehoused in a room with controlled temperature (23±1° C.), humidity(50±10%), and light (6:00 A.M. to 6:00 P.M.). Food and water wereavailable continuously. Animal care and use for experimental procedureswere approved by the Institutional Animal Care and Use Committee (IACUC)of Midwestern University. All anesthetic and surgical procedures were incompliance with the guidelines established by the IACUC of MidwesternUniversity.

Agents and Experimental Design

Amyloid-β (1-40) (Tocris Bioscience, Ellisville, Mo., USA), IRL-1620[N-Succinyl-[Glu9, Ala11,15] endothelin 1] (American Peptide Co, Inc.,Sunnyvale, Calif., USA) and BQ788 (American Peptide Co, Inc., Sunnyvale,Calif., USA) were dissolved in sterile saline and all the solutions werefreshly prepared before injections. Ketamine (Butler Animal HealthSupply, Dublin, Ohio, USA) was administered at a dose of 100 mg/kg,intraperitoneally (i.p.), and xylazine (Lloyd Laboratories, Shenandoah,Iowa, USA) was administered at a dose of 10 mg/kg, i.p. Rats wereanesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and alateral cerebral ventricle was cannulated by placing the rat in astereotaxic (David Kopf Instruments, Tujunga, Calif., USA) instrumentand fixing the cannula (coordinates: 1.0 mm lateral, 1.5 mm caudal tobregma and 4.0 mm deep from the bone). Cannula (Plastics One, Roanoke,Va., USA) was anchored with dental acrylic to three screws placed in theskull. The animals were allowed to recover from surgery for at leastseven days. After 7 days, rats were treated with vehicle, Aβ (1-40),ET_(B) receptor agonist and/or antagonist in the lateral cerebralventricles using the implanted cannula. Vehicle, Aβ or ET_(B) receptoragonist and antagonist were injected in a volume of 5 μl over a periodof 5 minutes. Aβ (1-40) was used because it is highly soluble comparedto Aβ (1-42) and induces endothelial dysfunction of both cerebral andsystemic blood vessels in addition to memory deficit (Nitta et al.,1994; Niwa et al., 2000; Smith et al., 2004; Weller et al., 1998).Agents were delivered using 10 μl Hamilton syringe and agent treatmentswere carried out individually using separate 10 μl syringe. IRL-1620 wasadministered 1 hour after Aβ injection whereas BQ788 was administered 15minutes prior to IRL-1620 injection. At the end of the experiment,placement of cannula was confirmed by injecting methylene blue dye (5μl) and observing the site and extent of staining. All experiments wereperformed on day 15 (Briyal et al., 2011). Water maze testing wasperformed from day 15 to day 19 after which animals were euthanized.Animals used for oxidative stress measurements were euthanized on day 15without being subjected to any behavioral testing. Diabetes mellitustype 2 was induced in rats belonging to the diabetic group byadministering freshly prepared streptozotocin at the dose of 45 mg/kg,i.p. Streptozotocin was dissolved in 0.01 M sodium citrate buffer of pH4.3. On day 3, blood glucose was obtained from the rat-tail and testedfor hyperglycemia using the SureStep Complete Blood Glucose monitor kit.Rats with blood glucose levels above 11.1 mM were considered diabetic.Diabetic and non-diabetic animals were randomly divided into five groups(6 rats per group) (i) Sham, (ii) Aβ+Vehicle, (iii) Aβ+IRL-1620, (iv)Aβ+BQ788 (v) Aβ+BQ788+IRL-1620. Aβ (1-40) was administeredintracerebroventricularly (i.c.v.) (20 μg in 3 equally divided dosesi.e. 6.67 μg were injected 3 times for a total of 20 μg dose) on day 1,7, and 14. Specific ET_(B) receptor agonist, IRL-1620 (3 μg) andspecific ET_(B) receptor antagonist, BQ788 (10 μg) were administeredi.c.v. daily for 14 days starting from day 1 of Aβ injection. The dosesof IRL-1620 and BQ788 were selected on the basis of previous studiesconducted in our laboratory (Leonard et al., 2011).

Estimation of Oxidative Stress Markers

Malondialdehyde (MDA), reduced glutathione (GSH) and superoxidedismutase (SOD) were estimated on day 15 in the rat brains. Rats weredecapitated and the brains quickly removed, cleaned with chilled salineand stored at −80° C. The biochemical analysis was performed within 48hours.

Measurement of Lipid Peroxidation

MDA, a marker of lipid peroxidation, was measured spectrophotometrically(Ohkawa et al., 1979). Briefly, the whole brain of each animal wasremoved separately and was homogenized with 10 times (w/v) in 0.1Msodium phosphate buffer (pH 7.4). Acetic acid 1.5 ml (20%), pH 3.5, 1.5ml thiobarbituric acid (0.8%) and 0.2 ml sodium dodecyl sulfate (8.1%)were added to 0.1 ml of processed tissue sample. The mixture was thenheated at 100° C. for 60 minutes. The mixture was cooled, and 5 ml ofbutanol:pyridine (15:1% v/v) and 1 ml of distilled water were added.After centrifugation at 4,000 rpm for 10 minutes, the organic layer waswithdrawn and absorbance was measured at 532 nm using aspectrophotometer.

Measurement of Glutathione

Glutathione was measured spectrophotometrically (Ellman, 1959). Briefly,whole brain was homogenized with 10 times (w/v) 0.1 M sodium phosphatebuffer (pH 7.4). This homogenate was then centrifuged with 5%trichloroacetic acid to separate the proteins. To 0.1 ml of supernatant,2 ml of phosphate buffer (pH 8.4), 0.5 ml of 5′5 dithiobis(2-nitrobenzoic acid) (DTNB) and 0.4 ml of double distilled water wasadded. The mixture was vortexed and the absorbance read at 412 nm within15 min.

Measurement of Superoxide Dismutase

SOD was estimated as described by Kakkar et al (Kakkar et al., 1984).Briefly, whole brain was homogenized with 10 times (w/v) 0.1 M sodiumphosphate buffer (pH 7.4). The reagents sodium pyrophosphate buffer 1.2ml (0.052 M) pH 8.3, 0.1 ml phenazine methosulphate (186 μM), 0.3 mlnitro blue tetrazolium (300 μM) and 0.2 ml NADH (780 μM) were added to0.1 ml of processed tissue sample. The mixture was then incubated for 90min at 30° C. Then 4 ml of n-butanol and 1 ml of acetic acid were added.The mixture was shaken vigorously. After centrifugation at 4,000 rpm for10 minutes, the organic layer was withdrawn and absorbance was measuredat 560 nm using a spectrophotometer. Protein was estimated using Lowry'smethod (Lowry et al., 1951).

Morris Water Maze (MWM) Test for Cognitive Impairment

Spatial learning and memory of animals were tested in a MWM (Morris,1984). A circular water tank (132 cm diameter, 60 cm height, paintedwhite was filled with water (25±2° C.) to a depth of 40 cm, the waterwas rendered opaque by the addition of non-fat milk. The pool wasdivided into four equal quadrants, labeled north, south, east, and west.A circular, white escape platform (10 cm in diameter) was submergedapproximately 2 cm below the surface of the water, 10 cm off the edge ofthe tank at a position designated as quadrant II (target quadrant). Avideo camera was mounted on the ceiling in the center of the pool. Theescapes latency and swimming path length was monitored with a Videomextracking system and data were collected using Videomex Water MazeSoftware (Columbus Instruments, Ohio, USA).

The platform remained in the same quadrant during the entire acquisitionphase experiments and removed in probe trial. Acquisition trials (4trials per day for 4 days) were started by placing rat in a pool facingthe wall of the tank from different randomly chosen start positions, andtime required to find the invisible platform was recorded. A triallasted until the rat found the platform or until 60 seconds had elapsedand an inter-trial interval of approximately 30 seconds. If rat did notfind the platform within 60 seconds, it was guided to the platform andplaced on it for 60 seconds. Time to reach the platform (latency inseconds) and swimming path length (in centimeters) was measured. Aftercompletion of the fourth trial on each day, the rat was dried andreturned to its home cage. Twenty four hours after the final acquisitiontrial, the platform was removed from the pool and a probe trial lasting60 seconds was performed; the time spent in the target quadrant wasrecorded. Time spent in the target quadrant indicated the degree ofmemory consolidation which had taken place after learning.

Statistical Analysis

Results were expressed as Mean±S.E.M. In acquisition trials of Morriswater maze following parameters were recorded: escape latency (timerequired to reach the platform from the releasing point in seconds), andpath length (distance traveled by rat from the release point to reachplatform in centimeters). Analysis of variance (ANOVA) was conducted onthese data, with group as the between-subject factor and with repeatedmeasures such as trial and day as within subject factors. Post hocanalysis (Tukey's test) was used to determine significance between thegroups. For probe trial data, time spent in quadrant II were recordedand analyzed by one way ANOVA and post hoc analysis by Bonferroni'stest. Oxidative stress measures were analyzed by one way ANOVA followedby post hoc analysis using Bonferroni's test. All analysis was carriedout using GraphPad Prism Statistical Software, version 5.00 (GraphPad,San Diego, Calif., USA). P<0.05 represents level of significance.

Effect of Diabetes Mellitus Type 2 on Rats

Diabetic rats were sluggish and had decreased locomotion as compared tonon-diabetic rats. However, diabetic and non-diabetic rats had similarperformance in Morris water maze tests (FIG. 8-11). FIG. 7 illustratesthat there was no difference in oxidative stress parameters betweendiabetic and non-diabetic rats.

Effect of ET_(B) Receptor Agonist and Antagonist on Oxidative StressParameters in Aβ-Treated Non-Diabetic and Diabetic Rats

To determine the involvement of ET_(B) receptors in Aβ induced changesin oxidative stress parameters, malondialdehyde, reduced glutathione andsuperoxide dismutase levels in the brains of sham and Aβ treated ratswere measured following administration of vehicle, IRL-1620 orBQ788+IRL-1620 (FIG. 1).

Effect on Brain Malondialdehyde Levels

Brain levels of malondialdehyde (MDA) were measured to determine theeffect of ET_(B) receptor stimulation on lipid peroxidation following Aβtreatment (FIG. 7A). As expected, levels of MDA were significantly(p<0.0001) higher in Aβ treated rats for both non-diabetic and diabeticgroups compared to sham groups. MDA level for non-diabetic Aβ treatedanimals was 516.13±14.02 nmol/g wet tissue which was greater compared tosham (112.1±1.84 nmol/g wet tissue) animals. In diabetic rats, MDA levelwas 531.58±9.02 nmol/g wet tissue in Aβ treated while it was 114.32±2.05nmol/g wet tissue in sham animals. MDA levels were significantly(P<0.001) reduced in IRL-1620 treated rats compared to vehicle treatedAβ rats for both non-diabetic (278.47±8.55 nmol/g wet tissue) anddiabetic (315.09±5.25 nmol/g wet tissue) animals. Administration ofBQ788 prior to IRL-1620, blocked IRL-1620 induced change in MDA levelsand the levels were similar to those seen in vehicle treated rats.

Effect on Brain Reduced Glutathione Levels

Reduced glutathione (GSH) levels in Aβ treated non-diabetic and diabeticanimals were significantly (P<0.0001) lower than those of sham operatedanimals. The mean GSH level for non-diabetic and diabetic Aβ treatedgroups were 102.5±5.96 and 81.2±4.33 μg/g wet tissue, respectively,while that in sham rats it was 239.1±8.0 μg/g wet tissue. Treatment withIRL-1620 significantly (P<0.001) increased levels of GSH in the brainsof Aβ treated non-diabetic and diabetic rats (192.74±6.26 and166.42±6.63 μg/g wet tissue, respectively) (FIG. 7B). Pretreatment withBQ788 blocked the positive effect of IRL-1620 treatment on GSH levels(81.2±5.49 μg/g wet tissue; P<0.001).

Effect on Brain Superoxide Dismutase Levels

The levels of superoxide dismutase (SOD) in the brains of vehicletreated non-diabetic (13.23±0.53 U/mg protein) and diabetic (11.07±0.54U/mg protein) Aβ rats were significantly (P<0.0001) lower than those ofsham operated group (35.22±1.43 U/mg protein). Administration ofIRL-1620 significantly improved SOD levels in non-diabetic and diabeticrats (22.26±1.16 and 21.4±1.65 U/mg protein, respectively) (FIG. 7C).Similar to GSH, SOD levels were significantly (P<0.001) lower whennon-diabetic and diabetic Aβ animals were pretreated with BQ788 prior toIRL-1620 administration (15.32±0.44 and 16.52±0.45 U/mg protein,respectively).

Effect of ET_(B) Receptor Agonist and Antagonist on Memory Deficit in AβTreated Non-Diabetic and Diabetic Rats

No significant difference was seen between non-diabetic and diabeticrats in water maze acquisition (FIGS. 8 and 10) and probe trial test(FIGS. 9 and 11). Rats treated with Aβ took significantly (p<0.0001)longer time (escape latency) to find the platform. Sham group improvedtheir performance in the hidden platform test as indicated by a decreasein escape latencies across successive days (day effect,F_((3,100))=3.968, p<0.001). There was a significant difference betweenescape latencies on day 1, 2, 3 and 4 in Aβ rats treated with vehiclecompared to sham (F_((3,59))=8.273, p<0.0001). However, when ET_(B)receptor agonist, IRL-1620 was administered to Aβ treated rats, escapelatency decreased significantly on day 3 and 4 of training when comparedto Aβ rats treated with vehicle (F_((4,59))=19.602, p<0.001). IRL-1620significantly improved cognitive impairment caused by Aβ treatment inrats. On the other hand, administration of an ET_(B) receptorantagonist, BQ788, blocked the improvement in escape latency produced byIRL-1620 in Aβ rats (FIGS. 8 and 10). A difference in escape latency isalso shown in representative trajectories of each group during anacquisition trial in the water maze task (FIGS. 8 and 10). The distancetraveled by a rat to reach the platform (path length) decreased acrossdays (F_((3,51))=76.3, p<0.0001), and there was also a significantday×group interaction (F_((12,100))=8.88, p<0.0001) indicatingdifferences among groups within each trial day. There was a significantdifference between sham, Aβ rats treated with vehicle and Aβ ratstreated with IRL-1620 (F_((2,57))=24.2, p<0.01); post hoc analysisshowed that Aβ rats treated with vehicle group swam longer path lengthsas compared to sham group (p<0.001). Aβ rats treated with IRL-1620 groupswam significantly (p<0.001) shorter path lengths as compared to vehiclegroup indicating beneficial effects of IRL-1620 treatment (FIGS. 8 and10). Aβ produced significant impairment of cognitive function in ratswhich could be improved with ET_(B) receptor agonist, and was blocked byspecific ET_(B) receptor antagonist.

Removal of platform from the target quadrant (quadrant II) resulted in ageneral tendency to swim preferentially in the target quadrant asopposed to other quadrants (probe trial). Therefore, time spent inquadrant II was compared for all the groups in order to observe effectof agents on memory retention. Time spent in other quadrants,specifically, does not reflect degree of memory consolidation and wastherefore not subjected to analysis. In probe trial, time spent in thetarget quadrant was significantly decreased in rats treated with Aβcompared to sham treated rats indicating memory deficit in Aβ treatedrats. Administration of ET_(B) receptor agonist, IRL1620, significantly(p<0.0001) increased time spent in the target quadrant compared tovehicle treated Aβ rats (FIGS. 9 and 11). Administration of an ET_(B)receptor antagonist, BQ788, to Aβ treated rats did not produce anyimprovement in time spent in the target quadrant. Difference in timespent in the target quadrant is shown in representative trajectories ofeach group during probe trial in the water maze task (FIGS. 9 and 11).Aβ produced significant memory deficit in rats which significantlyimproved with ET_(B) receptor agonist treatment but was blocked byspecific ET_(B) receptor antagonist.

The purpose of this study was to determine the effect of selectiveET_(B) receptor stimulation by IRL-1620 on functional recovery followingexperimentally induced cognitive impairment by intracerebroventicularinjections of Aβ in adult diabetic and non-diabetic rats. The currentfindings suggest that IRL-1620 produced a significant preventativeeffect in Aβ induced cognitive impairment in both diabetic andnon-diabetic rats. In order to confirm that the effects of IRL-1620 werespecific to stimulation of ET_(B) receptors, a selective ET_(B) receptorantagonist, BQ788, was used to block the effect of IRL-1620. Aβtreatment produced a significant increase in oxidative stress markers innon-diabetic and diabetic rats and treatment with selective ET_(B)receptor agonist, IRL-1620, significantly reduced oxidative stressmarkers. The reduction in oxidative stress marker induced by IRL-1620was blocked by pretreatment with specific ET_(B) receptor antagonist,BQ788, in Aβ non-diabetic and diabetic rats.

The incidence of diabetes mellitus and AD increases with age, and theincidence of AD is significantly higher in patients with diabetesmellitus (Janson et al., 2004). Pathologically, both are associated withaltered glucose homeostasis and extracellular accumulation of amyloidproteins. This suggests that there are common underlying mechanisms suchas cross-seeding of amyloid proteins or metabolic dysfunction.Therefore, the effect of IRL-1620 was studied in both non-diabetic anddiabetic rats treated with Aβ.

The prevalence of dementia, particularly of AD type is increasing and itis one of the most significant neurodegenerative disorders in theelderly. Recent studies support that metabolic and vascular dysfunctionsare involved in pathology and progression of AD. Vascular alterations,with impairment of glucose utilization and blood flow changes, occurwith and prior to AD diagnosis (Baquer et al., 2009; Casadesus et al.,2007; Meier-Ruge et al., 1994). These changes precede cognitiveimpairment and exacerbate underlying AD pathology. The discovery andprevention of vascular dysfunction could lead to new strategies toprevent or halt the progression of AD. Closely linked with vascularchanges in AD is Aβ, the major protein component of senile plaques in ADbrains. An elevated level of Aβ in the brain is one of the prominentfeatures of AD (Hardy and Selkoe, 2002). Tissues that produce the mostAβ are the brain and skeletal muscles, both of which possess highmetabolism and well-developed vascular networks (Cirrito et al., 2005;Ethell, 2010). Vascular damage and reactive gliosis are co-localizedwith amyloid deposits in AD brains, suggesting that vasculature may be aclinically significant site of AD pathology (Suo et al., 1998).

ET system has long been known to play an important role in theregulation of cerebral blood circulation. Several studies havedemonstrated involvement of ET in AD. Due to highly potentvasoconstriction caused by ET-1 via ET_(A) receptors, it was postulatedthat administration of an ET antagonist would decrease the damageassociated with AD. ET_(A) receptor antagonists, BQ123 and BMS182874,demonstrated reduced oxidative stress and improve the learning andmemory deficit following Aβ treatment in rats. However, a combined ETAIBreceptor antagonist had no beneficial effect (Briyal et al., 2011). Thisled us to investigate the involvement of ET_(B) receptors in AD. Infact, the ET binding sites in the brain are predominantly of ET_(B)receptors, and ET_(B) receptor agonists act as anti-apoptotic factoragainst the neurotoxicity of Aβ (Yagami et al., 2002). Elevation of Aβis directly implicated in vascular pathology, and vascular dysfunctionin AD is characterized by disruption of vascular architecture includinglower capillary density and reduced blood flow (Bell and Zlokovic, 2009;de la Torre, 1994; Iadecola et al., 2009; Zlokovic, 2008). Conversely,activation of endothelial ET_(B) receptors is known to elicitvasodilatation, and previous studies in our lab have indicated that thisleads to an increase in CBF in normal rats (Leonard and Gulati, 2009),indicating a possible role of ET_(B) agonists in the treatment andprevention of AD.

In the present study, Aβ treated rats received either vehicle orIRL-1620 for 14 days. On day 15, animals were evaluated for cognitiveimpairment and their brains were removed for analysis of oxidativestress markers. Treatment with IRL-1620 effectively reduced oxidativestress as measured by decreased levels of malondialdehyde (MDA) andincreased levels of reduced glutathione (GSH) and superoxide dismutase(SOD) compared to vehicle treated group. Blockade of ET_(B) receptorswith BQ788, on the other hand, resulted in an increase in oxidativestress. Increased levels of MDA along with decreased levels ofantioxidants GSH and SOD are all hallmarks of oxygen free radicalgeneration occurring as an early event in AD pathology (Cutler et al.,2004; Nunomura et al., 2001). The etiology of AD is thought to becomplex and initiates a variety of biochemical reactions leading toexcess intracellular Ca²⁺, glutamate excitotoxicity, production ofreactive oxygen species, and eventual apoptosis. Agents that targetthese events in order to slow or prevent irreversible injury are labeledas neuroprotective. Oxidative stress and Aβ are related to each other(Hensley et al., 1994; Mark et al., 1997). Oxidative stress alsocontributes to vascular dysfunction. It has been reported that oxidativedamage during pathogenesis of AD may be directly due to Aβ (Murray etal., 2007; Murray et al., 2005). It is disclosed herein that the ET_(B)receptor agonist, IRL-1620, decreased the oxidative stress markers thatwere increased by Aβ. It appears that Aβ can produce vasoconstrictionand an increase oxidative stress, both of which could be mediatedthrough ET. Stimulation of endothelial ET_(B) receptors is known toelicit vasodilatation, and previous studies in our lab have indicatedthat this leads to an increase in CBF. Hence, ET_(B) receptor agonistsmay be quite effective in preventing the damage due to Aβ in AD.

Thus, it is disclosed herein that stimulation of ET_(B) receptorsfollowing Aβ treatment leads to functional recovery. Behavioral studieswere conducted using MWM to determine whether ET_(B) receptor agonistsare able to improve the impairment of learning and memory caused by Aβ.It was found that Aβ produced a significant impairment in spatial memoryas evidenced by significantly longer escape latencies and no preferencefor the quadrant which previously contained the platform in the probetrial. Other researchers have also shown learning and memory deficitsdue to Aβ (Ahmed et al., 2010; Lopes et al., 2010; Tsukuda et al.,2009). It is shown herein that the specific ET_(B) receptor agonist,IRL-1620, significantly improved the spatial memory deficit caused by Aβtreatment. On the other hand, antagonism of ET_(B) receptors with BQ788,given prior to either vehicle or IRL-1620, resulted in learning andmemory deficit similar to those seen in vehicle group. These resultssuggest that the improvement seen with IRL-1620 is due to selectivestimulation of the ET_(B) receptors. The observed functional deficitscoincided with changes observed in oxidative stress markers.

Activation of ET_(B) receptors with IRL-1620 is known to cause cerebralvasodilatation and increased blood flow through release of nitric oxide(NO) (Kitazono et al., 1995; Leonard and Gulati, 2009; Tirapelli et al.,2005). Previous findings have revealed that cerebral neurovasculardysfunction in relation to bioavailability of NO formed by endothelialNO contributes to cognitive decline and neurodegeneration in AD (de laTorre et al., 2003). NO also plays an obligatory role in the regulationof CBF and cell viability and in the protection of nerve cells in AD(Toda et al., 2009). Recent studies have demonstrated that endothelialNO stimulation and increased CBF via pharmacological means enhanceangiogenesis (Chen et al., 2007; Ding et al., 2008). Along these lines,the ET_(B) receptor has been shown to enhance the formation of new bloodvessels through eNOS (Goligorsky et al., 1999). Previous reports usingan ET_(B) receptor deficient model have indicated that this receptorpromotes neuronal survival and decreases apoptosis in the hippocampus,dentate gyrus, and olfactory epithelium (Ehrenreich, 1999; Laziz et al.,2011; Riechers et al., 2004). Both induction of eNOS and directanti-apoptotic neuronal effects of ET_(B) receptor activation may play arole in reduction of oxidative stress and improvement in behavioralrecovery following AD.

In conclusion, reduction in oxidative stress and improvement incognitive impairment following ET_(B) receptor agonist, IRL-1620, andattenuation of these effects by a specific ET_(B) receptor antagonist,BQ788, in the current study indicates that ET_(B) receptors may be a newtherapeutic target for neuroprotection in AD.

Studies in Animal Model of Stroke

Effect of IRL-1620 on Levels of Brain Endothelin Receptors in MiddleCerebral Artery Occluded Rats:

ET_(B) receptors are present in large number in the CNS and appear toplay a key role in its development. It has been demonstrated that ET_(B)receptors in the brain are overexpressed at the time of birth and theirexpression decreases with maturity of the brain (Briyal et al., 2012b).It has also been shown that acute ischemic phase is followed by anintense sprouting of neurons and capillaries (Carmichael, 2006; Murphyand Corbett, 2009) along with activation of glial cells to create anenvironment for neuronal growth and plasticity (Hermann and Zechariah,2009; Zhang and Chopp, 2009). A regenerative response will bepharmacologically activated in the ischemic brain by stimulating ET_(B)receptors. Stimulation of ET_(B) receptors by IRL-1620 has been shown toprovide neuroprotective effect in MCAO rats (Leonard et al., 2011; 2012;Leonard and Gulati, 2013). ET_(A) receptors are increased in theinfarcted hemisphere at 24 hours post ischemia and subsequently returnto normal levels by one week, on the other hand a significant increasein ET_(B) receptor expression occurs after 1 week only in the infarctedhemisphere of rats treated with IRL-1620 (Leonard et al., 2012). It iscontemplated that IRL-1620 not only stimulates ET_(B) receptors but inlonger periods increases the number and affinity of these receptors.

Effect of IRL-1620 on Neurological Deficit Following Focal CerebralIschemia:

In a preliminary study, the effect of selectively activating ET_(B)receptors by IRL-1620 following permanent middle cerebral arteryocclusion in rats was determined. Twenty-four hours after middlecerebral artery occlusion, there was a significantly (P<0.001) higherneurological deficit and poor motor function compared to sham-operatedrats, indicative of neurological impairment following induction ofcerebral ischemia. Animals treated with IRL-1620 showed significantimprovement in all neurological and motor function tests when comparedwith vehicle-treated. In a longer term study, cerebral ischemia resultedin a distinct loss of motor coordination as measured by the foot faulterror and rota rod tests at 1, 4, and 7 days post infarction. Whereasvehicle-treated occluded rats performed worse with each assessment,animals treated with ET_(B) receptor agonist, IRL-1620, showed minimaldeficit at day one following occlusion, and improved over the course of7 days. Pretreatment with ET_(B) receptor antagonist, BQ-788, followedby either vehicle or IRL-1620 resulted in significantly more deficitsthan both sham-operated (P<0.001) or IRL-1620 (P<0.05) treatment,indicating that the improvement observed with IRL-1620 is specific tothe stimulation of ET_(B) receptors (Leonard et al., 2011; 2012).

Effect of IRL-1620 on Binding Characteristics of ET_(B) ReceptorsFollowing Focal Cerebral Ischemia:

Changes in binding characteristics of ET_(B) receptors were determinedin the brain, 1 and 7 days following MCAO. MCAO was produced in rats andbinding studies were performed using [¹²⁵I]-IRL-1620 (specific activity2200 Ci/mmol) as the radioligand and cold IRL-1620 (0-32 nM) asdisplacer. Non-specific binding was determined using 1 μM concentrationof IRL-1620. K_(d) and B_(max) values were calculated using GraphPadPrism version 5.00 for Windows (GraphPad Software, San Diego). Bindingcharacteristics (K_(d) and B_(max)) were not altered at 24 hours postMCAO. However, a significant decrease in K_(d) values of ET_(B) receptorbinding in both left and right hemispheres was observed 7 dayspost-MCAO. The decrease in K_(d) in the right (ischemic) hemisphere wassignificantly (P<0.001) greater compared to left (non-ischemic)hemisphere. B_(max) was increased in both left and right hemisphereswith the right hemisphere showing a significantly (P<0.001) greaterincrease compared to the left hemisphere (FIG. 14). It can be concludedthat an increase in the density and affinity of ET_(B) receptors on the7^(th) day of cerebral ischemia is an attempt to provide neuroprotectionof ischemic brain.

Effect IRL-1620 on Infarct Volume in Middle Cerebral Artery OccludedRats:

Middle cerebral artery occlusion for 7 days resulted in an infarctvolume of 177.06±13.21 mm³ in vehicle-treated rats. Administration ofIRL-1620 significantly reduced infarct volume (54.06±14.12 mm³; P<0.05)as compared with vehicle. Infarct volumes did not reduce when ET_(B)receptor antagonist, BQ-788, was given with either vehicle or IRL-1620(FIG. 12). A substantial edema was noted in the vehicle-treated animals,with the infarcted hemisphere 9.73±1.26% larger than the contralateralhemisphere, whereas IRL-1620-treated animals showed no significantedema, with infarcted hemisphere only 1.51±1.81% larger thannon-infarcted hemisphere. Conversely, blockade of the ET_(B) receptorwith BQ-788 followed by either vehicle or IRL-1620 treatmentsignificantly increased edema (17.02±3.17 and 17.97±5.17%, respectively,P<0.01) (Leonard et al., 2012).

Effect of IRL-1620 on 7 Day Survival of Rats Following Focal CerebralIschemia:

Studies were also conducted to determine the effect of stimulatingET_(B) receptors using a selective agonist, IRL-1620, in rats withmiddle cerebral artery occlusion (MCAO). It was found that there was nomortality in sham treated rats throughout the 7 day observation period.However, MCAO rats in the vehicle treated group presented with 38%mortality by 7th day. On the other hand, MCAO rats treated with IRL-1620showed no mortality throughout the 7 day period. However, MCAO ratstreated with an ET_(B) receptor antagonist, BQ-788+vehicle orBQ-788+IRL-1620, showed 25% mortality during the 7 day observation (FIG.13) (Leonard et al., 2012). Preliminary results and supportingliterature have prompted the investigation of the mechanism involved inneuroprotective and neurorestorative effects of stimulating ET_(B)receptors in rats with cerebral ischemia.

Effect of IRL-1620 on Angiogenesis and Neurogenesis Following CerebralIschemia in Rats:

Angiogenesis and neurogenesis are the driving forces for theneurovascular remodeling that is essential post-stroke to restore normalbrain function (Hawkins and Davis, 2005). VEGF is an endogenous proteinknown for its ability to promote angiogenesis and enhance vascularpermeability. Under hypoxic conditions such as cerebral ischemia, VEGFexpression is induced in neurons, astrocytes and endothelial cells viahypoxia-inducible factor-1 (HIF-1) (Breier and Risau, 1996). Onceexpressed, VEGF initiates both direct and indirect neuroprotectiveactions, inhibiting apoptosis, stimulating neurogenesis andangiogenesis, increasing glucose uptake and activating antioxidants(Gora-Kupilas and Josko, 2005). It has been shown thatintracerebroventricular (i.c.v.) administration of an ET_(B) receptoragonist in normal rats stimulates production of VEGF and activates VEGFreceptors in the brain, while, in cultured astrocytes, this agonistincreases VEGF-A mRNA as well as BrdU incorporation (Koyama et al.,2012; Koyama et al., 2011). ET_(B) receptor agonist, IRL-1620, has beenshown in our previous studies, to provide significant neuroprotection atboth 24 hours and 1 week following permanent cerebral ischemia.Therefore, the neuroprotective and neurorestorative effect followingstimulation of ET_(B) receptors in cerebral ischemic rats was studied(Leonard and Gulati, 2013). At 24 hours post occlusion, it was foundthat IRL-1620 treatment increased ET_(B) receptor expression andpreserved neuronal numbers in the cortex, striatum and subventricularzone (SVZ) of the ischemic rat brain (Figures. 15-22). IRL-1620 alsoenhanced the number of blood vessels labeled with vascular endothelialgrowth factor (VEGF) when compared to vehicle treatment (FIGS. 18-20).By 1 week following MCAO, VEGF-positive vessels/30 μm brain slice in theIRL-1620 group numbered 11.33±2.13 versus 4.19±0.79 in the vehicle group(P<0.01), indicating an increase in angiogenesis. Additionally, animalsreceiving IRL-1620 displayed an increased number of proliferating cells(P<0.0001) and cells positively staining for NGF (P<0.0001) in theinfarcted brain. NGF-positive cells in the cortex, striatum and SVZ ofIRL-1620 treated animals numbered 2.29±0.31, 2.08±0.26, and 3.05±0.38per 100 μm², respectively, demonstrating a significant increase inneurogenesis as compared to the vehicle group, which averaged less than1 NGF-positive cell per 100 μm² (FIGS. 21 and 22). Pretreatment withET_(B) antagonist, B Q-788, blocked the effects of IRL-1620 treatment,confirming the role of ET_(B) receptors in the neurovascular remodelingactions of IRL-1620. Results of the present study indicate thatIRL-1620, administered on the day of infarct, is neuroprotective andenhances angiogenic and neurogenic remodeling following cerebralischemia (Leonard and Gulati, 2013).

ETB Receptor Agonist, IRL-1620, in the Treatment of Ischemic Stroke:

It is disclosed herein that specific ETA receptor antagonists prevent Aβinduced increase in expression of ETA receptors, oxidative stress andcognitive deficits. However, it was observed that when a combined ETA/Breceptor antagonist was used, the beneficial effects were lost (Briyalet al., 2011). These findings led to the investigation of the role ofETB receptors in CNS disorders. ETB receptors are present in largenumber in the CNS and appear to play a key role in its development. Ithas been demonstrated that ETB receptors in the brain are overexpressedat the time of birth and their expression decreases with maturity of thebrain (Briyal et al., 2012b). It has also been shown that damaged brainexhibits a re-emergence of childhood organizational patterns,reminiscent of an ontogenetic state and is primed for recovery. However,endogenous remodeling of the CNS is not sufficient to restoreneurological function. It has been found that ET_(B) receptor agonist,IRL-1620 [Suc-[Glu9, Ala11,15]-Endothelin-1(8-12)], can increase theexpression of ET_(B) receptors in the CNS. An increase in ET_(B)receptors can produce reduction in apoptosis and promote angiogenesisand neurogenesis. It has been shown that expression of ET_(B) receptorsis increased in neurons, glia, and macrophages following ischemia.Additionally, studies demonstrate that ET_(B) receptor activationenhanced proliferation of neurons and inhibit apoptosis. A regenerativeresponse was pharmacologically activated in damaged brain by stimulatingETB receptors. ETB receptor stimulation, via selective ETB agonist,IRL-1620, significantly improved neurological deficit, motor functions,and oxidative stress markers and decreased infarct volume followingischemia in rats (Leonard et al., 2011; 2012). ETB receptor agonist,IRL-1620, provides significant neuroprotection at both 24 hours (Leonardet al., 2011) and 1 week (Leonard et al., 2012) following permanentcerebral ischemia and reduced infarct volume by 83.66% in acute studyand 69.49% in chronic study. IRL-1620 treatment increased ETB receptorexpression and preserved neuronal numbers in the cortex, striatum andsubventricular zone (SVZ) of the ischemic rat brain. IRL-1620 alsoenhanced the number of blood vessels labeled with vascular endothelialgrowth factor (VEGF) when compared to vehicle treatment (Leonard andGulati, 2013). Thus, IRL-1620 administered intravenously was found to behighly effective in preventing damage following stroke and aids in theneurovascular remodeling of ischemic brain by angiogenesis andneurogenesis (Leonard and Gulati, 2013). Studies further indicate thatstimulation of ETB receptors by IRL-1620 provides neuroprotection(Leonard et al., 2011; 2012), and it can be used as a therapeutic agentfor Alzheimer's disease (Briyal et al., 2011). It has been demonstratedthat IRL-1620 prevents cognitive impairment and oxidative stress inducedby Aβ (Briyal et al., 2011). It is contemplated that enhancement ofpossible survival mechanisms through stimulation of ET_(B) receptors byIRL-1620 leads to a better recovery following cerebral ischemia. Most ofthe stroke patients show substantial neurological improvement (Dimyanand Cohen, 2011) indicating endogenous restorative mechanisms. Hencethere is a potential to develop pharmacological agents that canstimulate and amplify these mechanisms. The two major approaches thatcan be used for the treatment of cerebral ischemia are neuroprotection,which requires an acute intervention, and neurorestoration, which can beinstituted during the stroke recovery phase (Andres et al., 2011;Bacigaluppi et al., 2009; Liu et al., 2008). Several trials have beenconducted or are in progress using pharmacological agents such asamphetamine, methylphenidate, levodopa, sildenafil, serotonin uptakeinhibitors, erythropoietin, statins, and granulocyte colony stimulatingfactor but none involves stimulation of ET_(B) receptors. It iscontemplated herein that stimulation of ET_(B) receptors produces, invarious embodiments, neuroprotection, neurorestoration, or both.

REFERENCES

-   Ahmed T, Enam S A and Gilani A H (2010) Curcuminoids enhance memory    in an amyloid-infused rat model of Alzheimer's disease. Neuroscience    169:1296-1306.-   Andres R H, Horie N, Slikker W, Keren-Gill H, Zhan K, Sun G, Manley    N C, Pereira M P, Sheikh L A, McMillan E L, Schaar B T, Svendsen C    N, Bliss T M and Steinberg G K (2011) Human neural stem cells    enhance structural plasticity and axonal transport in the ischaemic    brain. Brain: a journal of neurology 134:1777-1789.-   Asano T, Ikegaki I, Satoh S, Suzuki Y, Shibuya M, Sugita K and    Hidaka H (1990) Endothelin: a potential modulator of cerebral    vasospasm. European journal of pharmacology 190:365-372.-   Bacigaluppi M, Pluchino S, Peruzzotti-Jametti L, Kilic E, Kilic U,    Salani G, Brambilla E, West M I,-   Comi G, Martino G and Hermann D M (2009) Delayed post-ischaemic    neuroprotection following systemic neural stem cell transplantation    involves multiple mechanisms. Brain: a journal of neurology    132:2239-2251.-   Baquer N Z, Taha A, Kumar P, McLean P, Cowsik S M, Kale R K, Singh R    and Sharma D (2009) A metabolic and functional overview of brain    aging linked to neurological disorders. Bio gerontology 10:377-413.-   Barone F C, Ohlstein E H, Hunter A J, Campbell Calif., Hadingham S    H, Parsons A A, Yang Y and Shohami E (2000) Selective antagonism of    endothelin-A-receptors improves outcome in both head trauma and    focal stroke in rat. Journal of cardiovascular pharmacology    36:S357-361.-   Barone F C, White R F, Elliott J D, Feuerstein G Z and Ohlstein E    H (1995) The endothelin receptor antagonist S B 217242 reduces    cerebral focal ischemic brain injury. Journal of cardiovascular    pharmacology 26 Suppl 3:S404-407.-   Bath P M and Lees K R (2000) ABC of arterial and venous disease.    Acute stroke. BMJ 320:920-923.-   Bell R D and Zlokovic B V (2009) Neurovascular mechanisms and    blood-brain barrier disorder in Alzheimer's disease. Acta    neuropathologica 118:103-113.-   Bredesen D E, Rao R V and Mehlen P (2006) Cell death in the nervous    system. Nature 443:796-802.-   Breier G and Risau W (1996) The role of vascular endothelial growth    factor in blood vessel formation. Trends in cell biology 6:454-456.-   Briyal S and Gulati A (2010) Endothelin-A receptor antagonist BQ123    potentiates acetaminophen induced hypothermia and reduces infarction    following focal cerebral ischemia in rats. European journal of    pharmacology 644:73-79.-   Briyal S, Gulati A and Gupta Y K (2007) Effect of combination of    endothelin receptor antagonist (TAK-044) and aspirin in middle    cerebral artery occlusion model of acute ischemic stroke in rats.    Methods Find Exp Clin Pharmacol 29:257-263.-   Briyal S, Gulati K and Gulati A (2012a) Repeated administration of    exendin-4 reduces focal cerebral ischemia-induced infarction in    rats. Brain research 1427:23-34.-   Briyal S, Lavhale M S and Gulati A (2012b) Repeated administration    of centhaquin to pregnant rats did not affect postnatal development    and expression of endothelin receptors in the brain, heart or kidney    of pups. Arzneimittel-Forschung 62:670-676.-   Briyal S, Philip T and Gulati A (2011) Endothelin-A receptor    antagonists prevent amyloid-beta-induced increase in ETA receptor    expression, oxidative stress, and cognitive impairment. Journal of    Alzheimer's disease: TAD 23:491-503.-   Carmichael S T (2006) Cellular and molecular mechanisms of neural    repair after stroke-making waves. Annals of neurology 59:735-742.-   Casadesus G, Moreira P I, Nunomura A, Siedlak S L, Bligh-Glover W,    Balraj E, Petot G, Smith M A and Perry G (2007) Indices of metabolic    dysfunction and oxidative stress. Neurochemical research 32:717-722.-   Chen J, Cui X, Zacharek A, Jiang H, Roberts C, Zhang C, Lu M, Kapke    A, Feldkamp C S and Chopp M (2007) Niaspan increases angiogenesis    and improves functional recovery after stroke. Annals of neurology    62:49-58.-   Chuquet J, Benchenane K, Toutain J, MacKenzie E T, Roussel S and    Touzani 0 (2002) Selective blockade of endothelin-B receptors    exacerbates ischemic brain damage in the rat. Stroke; a journal of    cerebral circulation 33:3019-3025.-   Cirrito J R, Yamada K A, Finn M B, Sloviter R S, Bales K R, May P C,    Schoepp D D, Paul S M, Mennerick S and Holtzman D M (2005) Synaptic    activity regulates interstitial fluid amyloid-beta levels in vivo.    Neuron 48:913-922.-   Cutler R G, Kelly J, Storie K, Pedersen W A, Tammara A, Hatanpaa K,    Troncoso J C and Mattson M P (2004) Involvement of oxidative    stress-induced abnormalities in ceramide and cholesterol metabolism    in brain aging and Alzheimer's disease. Proceedings of the National    Academy of Sciences of the United States of America 101:2070-2075.-   de la Torre J C (1994) Impaired brain microcirculation may trigger    Alzheimer's disease. Neuroscience and biobehavioral reviews    18:397-401.-   de la Torre J C, Pappas B A, Prevot V, Emmerling M R, Mantione K,    Fortin T, Watson M D and Stefano G B (2003) Hippocampal nitric oxide    upregulation precedes memory loss and A beta 1-40 accumulation after    chronic brain hypoperfusion in rats. Neurological research    25:635-641.-   Deb P, Sharma S and Hassan K M (2010) Pathophysiologic mechanisms of    acute ischemic stroke: An overview with emphasis on therapeutic    significance beyond thrombolysis. Pathophysiology 17:197-218.-   Dembowski C, Hofmann P, Koch T, Kamrowski-Kruck H, Riedesel H,    Krammer H J, Kaup F J and Ehrenreich H (2000) Phenotype, intestinal    morphology, and survival of homozygous and heterozygous endothelin B    receptor—deficient (spotting lethal) rats. J Pediatr Surg    35:480-488.-   Dimyan M A and Cohen L G (2011) Neuroplasticity in the context of    motor rehabilitation after stroke. Nature reviews Neurology 7:76-85.-   Ding G, Jiang Q, Li L, Zhang L, Zhang Z G, Ledbetter K A, Panda S,    Davarani S P, Athiraman H, Li Q, Ewing J R and Chopp M (2008)    Magnetic resonance imaging investigation of axonal remodeling and    angiogenesis after embolic stroke in sildenafil-treated rats.    Journal of cerebral blood flow and metabolism: official journal of    the International Society of Cerebral Blood Flow and Metabolism    28:1440-1448.-   Donnan G A, Fisher M, Macleod M and Davis S M (2008) Stroke. Lancet    371:1612-1623.-   Ehrenreich H (1999) The astrocytic endothelin system: toward solving    a mystery focus on “distinct pharmacological properties of ET-1 and    ET-3 on astroglial gap junctions and Ca(2+) signaling”. The American    journal of physiology 277:C614-615.-   Ehrenreich H, Nau T R, Dembowski C, Hasselblatt M, Barth M, Hahn A,    Schilling L, Siren A L and Bruck W (2000) Endothelin b receptor    deficiency is associated with an increased rate of neuronal    apoptosis in the dentate gyrus. Neuroscience 95:993-1001.-   Ehrenreich H, Oldenburg J, Hasselblatt M, Herms J, Dembowski C,    Loffler B M, Bruck W, Kamrowski-Kruck H, Gall S, Siren A L and    Schilling L (1999) Endothelin B receptor-deficient rats as a    subtraction model to study the cerebral endothelin system.    Neuroscience 91:1067-1075.-   Ellman G L (1959) Tissue sulfhydryl groups. Archives of biochemistry    and biophysics 82:70-77.-   Ethell D W (2010) An amyloid-notch hypothesis for Alzheimer's    disease. The Neuroscientist: a review journal bringing neurobiology,    neurology and psychiatry 16:614-617.-   Feigin V L, Lawes C M, Bennett D A, Barker-Collo S L and Parag    V (2009) Worldwide stroke incidence and early case fatality reported    in 56 population-based studies: a systematic review. Lancet Neurol    8:355-369.-   Fisher M and Norrving B (2011) The International Agenda for Stroke,    in 1st Global Conference on Healthy Lifestyles and Noncommunicable    Diseases Control (Association A H ed), American Heart Association,    Moscow.-   Font M A, Arboix A and Krupinski J (2010) Angiogenesis, neurogenesis    and neuroplasticity in ischemic stroke. Current cardiology reviews    6:238-244.-   Gil-Mohapel J, Boehme F, Kainer L and Christie B R (2010)    Hippocampal cell loss and neurogenesis after fetal alcohol exposure:    insights from different rodent models. Brain Res Rev 64:283-303.-   Goligorsky M S, Budzikowski A S, Tsukahara H and Noiri E (1999)    Co-operation between endothelin and nitric oxide in promoting    endothelial cell migration and angiogenesis. Clinical and    experimental pharmacology & physiology 26:269-271.-   Gora-Kupilas K and Josko J (2005) The neuroprotective function of    vascular endothelial growth factor (VEGF). Folia    neuropathologica/Association of Polish Neuropathologists and Medical    Research Centre, Polish Academy of Sciences 43:31-39.-   Goto K, Kasuya Y, Matsuki N, Takuwa Y, Kurihara H, Ishikawa T,    Kimura S, Yanagisawa M and Masaki T (1989) Endothelin activates the    dihydropyridine-sensitive, voltage-dependent Ca2+ channel in    vascular smooth muscle. Proceedings of the National Academy of    Sciences of the United States of America 86:3915-3918.-   Gulati A, Kumar A, Morrison S and Shahani B T (1997) Effect of    centrally administered endothelin agonists on systemic and regional    blood circulation in the rat: role of sympathetic nervous system.    Neuropeptides 31:301-309.-   Gulati A, Kumar A and Shahani B T (1996) Cardiovascular effects of    centrally administered endothelin-1 and its relationship to changes    in cerebral blood flow. Life sciences 58:437-445.-   Gulati A, Rebello S, Roy S and Saxena P R (1995) Cardiovascular    effects of centrally administered endothelin-1 in rats. Journal of    cardiovascular pharmacology 26 Suppl 3:S244-246.-   Gupta Y K, Briyal S, Sharma U, Jagannathan N R and Gulati A (2005)    Effect of endothelin antagonist (TAK-044) on cerebral ischemic    volume, oxidative stress markers and neurobehavioral parameters in    the middle cerebral artery occlusion model of stroke in rats. Life    sciences 77:15-27.-   Han B H, Zhou M L, Abousaleh F, Brendza R P, Dietrich H H,    Koenigsknecht-Talboo J, Cirrito J R, Milner E, Holtzman D M and    Zipfel G J (2008) Cerebrovascular dysfunction in amyloid precursor    protein transgenic mice: contribution of soluble and insoluble    amyloid-beta peptide, partial restoration via gamma-secretase    inhibition. The Journal of neuroscience: the official journal of the    Society for Neuroscience 28:13542-13550.-   Hardy J and Selkoe D J (2002) The amyloid hypothesis of Alzheimer's    disease: progress and problems on the road to therapeutics. Science    297:353-356.-   Hawkins B T and Davis T P (2005) The blood-brain    barrier/neurovascular unit in health and disease. Pharmacological    reviews 57:173-185.-   Hensley K, Carney J M, Mattson M P, Aksenova M, Harris M, Wu J F,    Floyd R A and Butterfield D A (1994) A model for beta-amyloid    aggregation and neurotoxicity based on free radical generation by    the peptide: relevance to Alzheimer disease. Proceedings of the    National Academy of Sciences of the United States of America    91:3270-3274.-   Hermann D M and Zechariah A (2009) Implications of vascular    endothelial growth factor for postischemic neurovascular remodeling.    Journal of cerebral blood flow and metabolism: official journal of    the International Society of Cerebral Blood Flow and Metabolism    29:1620-1643.-   Hoehn B D, Harik S I and Hudetz A G (2002) VEGF mRNA expressed in    microvessels of neonatal and adult rat cerebral cortex. Brain Res    Mol Brain Res 101:103-108.-   Iadecola C, Park L and Capone C (2009) Threats to the mind: aging,    amyloid, and hypertension. Stroke; a journal of cerebral circulation    40:S40-44.-   Janson J, Laedtke T, Parisi J E, O'Brien P, Petersen R C and Butler    P C (2004) Increased risk of type 2 diabetes in Alzheimer disease.    Diabetes 53:474-481.-   Johnson D K, Storandt M, Morris J C, Langford Z D and Galvin J    E (2008) Cognitive profiles in dementia: Alzheimer disease vs    healthy brain aging. Neurology 71:1783-1789.-   Kakkar P, Das B and Viswanathan P N (1984) A modified    spectrophotometric assay of superoxide dismutase. Indian journal of    biochemistry & biophysics 21:130-132.-   Kaundal R K, Deshpande T A, Gulati A and Sharma S S (2012) Targeting    endothelin receptors for pharmacotherapy of ischemic stroke: current    scenario and future perspectives. Drug Discov Today 17:793-804.-   Kitazono T, Heistad D D and Faraci F M (1995) Enhanced responses of    the basilar artery to activation of endothelin-B receptors in    stroke-prone spontaneously hypertensive rats. Hypertension    25:490-494.-   Kohzuki M, Onodera H, Yasujima M, Itoyama Y, Kanazawa M, Sato T and    Abe K (1995) Endothelin receptors in ischemic rat brain and    Alzheimer brain. Journal of cardiovascular pharmacology 26 Suppl    3:S329-331.-   Kojima T, Isozaki-Fukuda Y, Takedatsu M, Hirata Y and Kobayashi    Y (1992) Circulating levels of endothelin and atrial natriuretic    factor during postnatal life. Acta Paediatr 81:676-677.-   Koyama Y, Maebara Y, Hayashi M, Nagae R, Tokuyama S and Michinaga    S (2012) Endothelins reciprocally regulate VEGF-A and angiopoietin-1    production in cultured rat astrocytes: implications on astrocytic    proliferation. Glia 60:1954-1963.-   Koyama Y, Nagae R, Tokuyama S and Tanaka K (2011) I.c.v    administration of an endothelin ET(B) receptor agonist stimulates    vascular endothelial growth factor-A production and activates    vascular endothelial growth factor receptors in rat brain.    Neuroscience 192:689-698.-   Laziz I, Larbi A, Grebert D, Sautel M, Congar P, Lacroix M C,    Salesse R and Meunier N (2011) Endothelin as a neuroprotective    factor in the olfactory epithelium. Neuroscience 172:20-29.-   Lee H O, Levorse J M and Shin M K (2003) The endothelin receptor-B    is required for the migration of neural crest-derived melanocyte and    enteric neuron precursors. Dev Biol 259:162-175.-   Leonard M G, Briyal S and Gulati A (2011) Endothelin B receptor    agonist, IRL-1620, reduces neurological damage following permanent    middle cerebral artery occlusion in rats. Brain research 1420:48-58.-   Leonard M G, Briyal S and Gulati A (2012) Endothelin B receptor    agonist, IRL-1620, provides long-term neuroprotection in cerebral    ischemia in rats. Brain research 1464:14-23.-   Leonard M G and Gulati A (2009) Repeated administration of ET(B)    receptor agonist, IRL-1620, produces tachyphylaxis only to its    hypotensive effect. Pharmacological research: the official journal    of the Italian Pharmacological Society 60:402-410.-   Leonard M G and Gulati A (2013) Endothelin B receptor agonist,    IRL-1620, enhances angiogenesis and neurogenesis following cerebral    ischemia in rats. Brain research 1528:28-41.-   Levin E R (1995) Endothelins. The New England journal of medicine    333:356-363.-   Li L, Xiong Y, Qu Y, Mao M, Mu W, Wang H and Mu D (2008) The    requirement of extracellular signal-related protein kinase pathway    in the activation of hypoxia inducible factor 1 alpha in the    developing rat brain after hypoxia-ischemia. Acta neuropathologica    115:297-303.-   Liu Z, Li Y, Zhang X, Savant-Bhonsale S and Chopp M (2008)    Contralesional axonal remodeling of the corticospinal system in    adult rats after stroke and bone marrow stromal cell treatment.    Stroke; a journal of cerebral circulation 39:2571-2577.-   Loo L S, Ng Y K, Zhu Y Z, Lee H S and Wong P T (2002) Cortical    expression of endothelin receptor subtypes A and B following middle    cerebral artery occlusion in rats. Neuroscience 112:993-1000.-   Lopes J P, Oliveira C R and Agostinho P (2010) Neurodegeneration in    an Abeta-induced model of Alzheimer's disease: the role of Cdk5.    Aging cell 9:64-77.-   Lowry O H, Rosebrough N J, Farr A L and Randall R J (1951) Protein    measurement with the Folin phenol reagent. The Journal of biological    chemistry 193:265-275.-   Ly J V, Zavala J A and Donnan G A (2006) Neuroprotection and    thrombolysis: combination therapy in acute ischaemic stroke. Expert    Opin Pharmacother 7:1571-1581.-   Malik S, Vinukonda G, Vose L R, Diamond D, Bhimavarapu B B, Hu F,    Zia M T, Hevner R, Zecevic N and Ballabh P (2013) Neurogenesis    continues in the third trimester of pregnancy and is suppressed by    premature birth. The Journal of neuroscience: the official journal    of the Society for Neuroscience 33:411-423.-   Mark R J, Lovell M A, Markesbery W R, Uchida K and Mattson M    P (1997) A role for 4-hydroxynonenal, an aldehydic product of lipid    peroxidation, in disruption of ion homeostasis and neuronal death    induced by amyloid beta-peptide. Journal of neurochemistry    68:255-264.-   Mathers C D, Boerma T and Ma Fat D (2009) Global and regional causes    of death. Br Med Bull 92:7-32.-   Meier-Ruge W, Bertoni-Freddari C and Iwangoff P (1994) Changes in    brain glucose metabolism as a key to the pathogenesis of Alzheimer's    disease. Gerontology 40:246-252.-   Micieli G, Marcheselli S and Tosi P A (2009) Safety and efficacy of    alteplase in the treatment of acute ischemic stroke. Vasc Health    Risk Manag 5:397-409.-   Minami M, Kimura M, Iwamoto N and Arai H (1995) Endothelin-1-like    immunoreactivity in cerebral cortex of Alzheimer-type dementia.    Progress in neuro-psychopharmacology & biological psychiatry    19:509-513.-   Morris R (1984) Developments of a water-maze procedure for studying    spatial learning in the rat. Journal of neuroscience methods    11:47-60.-   Murphy T H and Corbett D (2009) Plasticity during stroke recovery:    from synapse to behaviour. Nature reviews Neuroscience 10:861-872.-   Murray I V, Liu L, Komatsu H, Uryu K, Xiao G, Lawson J A and Axelsen    P H (2007) Membrane-mediated amyloidogenesis and the promotion of    oxidative lipid damage by amyloid beta proteins. The Journal of    biological chemistry 282:9335-9345.-   Murray I V, Sindoni M E and Axelsen P H (2005) Promotion of    oxidative lipid membrane damage by amyloid beta proteins.    Biochemistry 44:12606-12613.-   Nitta A, Itoh A, Hasegawa T and Nabeshima T (1994) beta-Amyloid    protein-induced Alzheimer's disease animal model. Neuroscience    letters 170:63-66.-   Niwa K, Carlson G A and ladecola C (2000) Exogenous A beta1-40    reproduces cerebrovascular alterations resulting from amyloid    precursor protein overexpression in mice. Journal of cerebral blood    flow and metabolism: official journal of the International Society    of Cerebral Blood Flow and Metabolism 20:1659-1668.-   Niwa K, Kazama K, Younkin L, Younkin S G, Carlson G A and ladecola    C (2002) Cerebrovascular autoregulation is profoundly impaired in    mice overexpressing amyloid precursor protein. American journal of    physiology Heart and circulatory physiology 283:H315-323.-   Niwa K, Porter V A, Kazama K, Cornfield D, Carlson G A and Iadecola    C (2001) A beta-peptides enhance vasoconstriction in cerebral    circulation. American journal of physiology Heart and circulatory    physiology 281:H2417-2424.-   Nowacka M M and Obuchowicz E (2012) Vascular endothelial growth    factor (VEGF) and its role in the central nervous system: a new    element in the neurotrophic hypothesis of antidepressant drug    action. Neuropeptides 46:1-10.-   Nunomura A, Perry G, Aliev G, Hirai K, Takeda A, Balraj E K, Jones P    K, Ghanbari H, Wataya T, Shimohama S, Chiba S, Atwood C S, Petersen    R B and Smith M A (2001) Oxidative damage is the earliest event in    Alzheimer disease. Journal of neuropathology and experimental    neurology 60:759-767.-   Ogunshola 00, Stewart W B, Mihalcik V, Solli T, Madri J A and Ment L    R (2000) Neuronal VEGF expression correlates with angiogenesis in    postnatal developing rat brain. Brain research Developmental brain    research 119:139-153.-   Ohkawa H, Ohishi N and Yagi K (1979) Assay for lipid peroxides in    animal tissues by thiobarbituric acid reaction. Analytical    biochemistry 95:351-358.-   Paris D, Quadros A, Humphrey J, Patel N, Crescentini R, Crawford F    and Mullan M (2004) Nilvadipine antagonizes both Abeta vasoactivity    in isolated arteries, and the reduced cerebral blood flow in APPsw    transgenic mice. Brain research 999:53-61.-   Patel T R, Galbraith S L, McAuley M A, Doherty A M, Graham D I and    McCulloch J (1995) Therapeutic potential of endothelin receptor    antagonists in experimental stroke. Journal of cardiovascular    pharmacology 26 Suppl 3:S412-415.-   Quinn R (2005) Comparing rat's to human's age: how old is my rat in    people years? Nutrition 21:775-777.-   Rebello S, Roy S, Saxena P R and Gulati A (1995a) Systemic    hemodynamic and regional circulatory effects of centrally    administered endothelin-1 are mediated through ETA receptors. Brain    research 676:141-150.-   Rebello S, Singh G and Gulati A (1995b) Elevated levels of    endothelin-1 following unilateral cerebral-ischemia in rats. Faseb    Journal 9:A937-A.-   Riechers C C, Knabe W, Siren A L, Gariepy C E, Yanagisawa M and    Ehrenreich H (2004) Endothelin B receptor deficient transgenic    rescue rats: a rescue phenomenon in the brain. Neuroscience    124:719-723.-   Roger V L, Go A S, Lloyd-Jones D M, Benjamin E J, Berry J D, Borden    W B, Bravata D M, Dai S, Ford E S, Fox C S, Fullerton H J, Gillespie    C, Hailpern S M, Heit J A, Howard V J, Kissela B M, Kittner S J,    Lackland D T, Lichtman J H, Lisabeth L D, Makuc D M, Marcus G M,    Marelli A, Matchar D B, Moy C S, Mozaffarian D, Mussolino M E,    Nichol G, Paynter N P, Soliman E Z, Sorlie P D, Sotoodehnia N, Turan    T N, Virani S S, Wong N D, Woo D and Turner M B (2012) Heart disease    and stroke statistics—2012 update: a report from the American Heart    Association. Circulation 125:e2-e220.-   Rosenstein J M, Krum J M and Ruhrberg C (2010) VEGF in the nervous    system. Organogenesis 6:107-114.-   Rubinsztein D C (2006) The roles of intracellular    protein-degradation pathways in neurodegeneration. Nature    443:780-786.-   Schiffrin E L, Intengan H D, Thibault G and Touyz R M (1997)    Clinical significance of endothelin in cardiovascular disease. Curr    Opin Cardiol 12:354-367.-   Schinelli S (2006) Pharmacology and physiopathology of the brain    endothelin system: an overview. Curr Med Chem 13:627-638.-   Schneider M P, Boesen E I and Pollock D M (2007) Contrasting actions    of endothelin ET(A) and ET(B) receptors in cardiovascular disease.    Annu Rev Pharmacol Toxicol 47:731-759.-   Shin H K, Jones P B, Garcia-Alloza M, Borrelli L, Greenberg S M,    Bacskai B J, Frosch M P, Hyman B T, Moskowitz M A and Ayata C (2007)    Age-dependent cerebrovascular dysfunction in a transgenic mouse    model of cerebral amyloid angiopathy. Brain: a journal of neurology    130:2310-2319.-   Sims N R and Muyderman H (2009) Mitochondria, oxidative metabolism    and cell death in stroke, in Biochimica et biophysica acta pp 80-91.-   Smith C C, Stanyer L and Betteridge D J (2004) Soluble beta-amyloid    (A beta) 40 causes attenuation or potentiation of    noradrenaline-induced vasoconstriction in rats depending upon the    concentration employed. Neuroscience letters 367:129-132.-   Steinwachs D M, Collins-Nakai R L, Cohn L H, Garson A, Jr. and Wolk    M J (2000) The future of cardiology: utilization and costs of care.    J Am Coll Cardiol 35:91B-98B.-   Strong K, Mathers C and Bonita R (2007) Preventing stroke: saving    lives around the world. Lancet Neurol 6:182-187.-   Suo Z, Humphrey J, Kundtz A, Sethi F, Placzek A, Crawford F and    Mullan M (1998) Soluble Alzheimers beta-amyloid constricts the    cerebral vasculature in vivo. Neuroscience letters 257:77-80.-   Tirapelli C R, Casolari D A, Yogi A, Montezano A C, Tostes R C,    Legros E, D'Orleans-Juste P and de Oliveira A M (2005) Functional    characterization and expression of endothelin receptors in rat    carotid artery: involvement of nitric oxide, a vasodilator    prostanoid and the opening of K+ channels in ETB-induced relaxation.    British journal of pharmacology 146:903-912.-   Toda N, Ayajiki K and Okamura T (2009) Cerebral blood flow    regulation by nitric oxide: recent advances. Pharmacological reviews    61:62-97.-   Trollmann R, Schneider J, Keller S, Strasser K, Wenzel D, Rascher W,    Ogunshola 00 and Gassmann M (2008) HIF-1-regulated vasoactive    systems are differentially involved in acute hypoxic stress    responses of the developing brain of newborn mice and are not    affected by levetiracetam. Brain research 1199:27-36.-   Tsukahara H, Ende H, Magazine H I, Bahou W F and Goligorsky M    S (1994) Molecular and functional characterization of the    non-isopeptide-selective ETB receptor in endothelial cells. Receptor    coupling to nitric oxide synthase. The Journal of biological    chemistry 269:21778-21785.-   Tsukuda K, Mogi M, Iwanami J, Min L J, Sakata A, Jing F, Iwai M and    Horiuchi M (2009) Cognitive deficit in amyloid-beta-injected mice    was improved by pretreatment with a low dose of telmisartan partly    because of peroxisome proliferator-activated receptor-gamma    activation. Hypertension 54:782-787.-   Vidovic M, Chen M M, Lu Q Y, Kalloniatis K F, Martin B M, Tan A H,    Lynch C, Croaker G D, Cass D T and Song Z M (2008) Deficiency in    endothelin receptor B reduces proliferation of neuronal progenitors    and increases apoptosis in postnatal rat cerebellum. Cellular and    molecular neurobiology 28:1129-1138.-   Viossat I, Duverger D, Chapelat M, Pirotzky E, Chabrier P E and    Braquet P (1993) Elevated tissue endothelin content during focal    cerebral ischemia in the rat. Journal of cardiovascular pharmacology    22 Suppl 8:S306-309.-   Virgintino D, Errede M, Robertson D, Girolamo F, Masciandaro A and    Bertossi M (2003) VEGF expression is developmentally regulated    during human brain angiogenesis. Histochem Cell Biol 119:227-232.-   Weller R O, Massey A, Newman T A, Hutchings M, Kuo Y M and Roher A    E (1998) Cerebral amyloid angiopathy: amyloid beta accumulates in    putative interstitial fluid drainage pathways in Alzheimer's    disease. The American journal of pathology 153:725-733.-   Yagami T, Ueda K, Asakura K, Kuroda T, Hata S, Sakaeda T, Kambayashi    Y and Fujimoto M (2002) Effects of endothelin B receptor agonists on    amyloid beta protein (25-35)-induced neuronal cell death. Brain    research 948:72-81.-   Yagami T, Ueda K, Sakaeda T, Okamura N, Nakazato H, Kuroda T, Hata    S, Sakaguchi G, Itoh N, Hashimoto Y and Fujimoto M (2005) Effects of    an endothelin B receptor agonist on secretory phospholipase    A2-IIA-induced apoptosis in cortical neurons. Neuropharmacology    48:291-300.-   Yoshizawa T, Iwamoto H, Mizusawa H, Suzuki N, Matsumoto H and    Kanazawa I (1992) Cerebrospinal fluid endothelin-1 in Alzheimer's    disease and senile dementia of Alzheimer type. Neuropeptides    22:85-88.-   Zhang R L, Zhang C, Zhang L, Roberts C, Lu M, Kapke A, Cui Y,    Ninomiya M, Nagafuji T, Albala B, Zhang Z G and Chopp M (2008)    Synergistic effect of an endothelin type A receptor antagonist,    5-0139, with rtPA on the neuroprotection after embolic stroke.    Stroke; a journal of cerebral circulation 39:2830-2836.-   Zhang W W, Badonic T, Hoog A, Jiang M H, Ma K C, Nie X J and Olsson    Y (1994) Astrocytes in Alzheimer's disease express immunoreactivity    to the vaso-constrictor endothelin-1. Journal of the neurological    sciences 122:90-96.-   Zhang Y, Belayev L, Zhao W, Irving E A, Busto R and Ginsberg M    D (2005) A selective endothelin ET(A) receptor antagonist, S B    234551, improves cerebral perfusion following permanent focal    cerebral ischemia in rats. Brain research 1045:150-156.-   Zhang Z G and Chopp M (2009) Neurorestorative therapies for stroke:    underlying mechanisms and translation to the clinic. Lancet Neurol    8:491-500.-   Zlokovic B V (2008) New therapeutic targets in the neurovascular    pathway in Alzheimer's disease. Neurotherapeutics: the journal of    the American Society for Experimental Neuro Therapeutics 5:409-414.

TABLE 1 Effect of ET_(B) receptor agonist, IRL-1620, and antagonist,BQ788, on neurological deficit and motor function post middle cerebralartery occlusion. IRL-1620 (5 μg/kg, i.v.) or isotonic saline (1 ml/kg,i.v.) was injected at 2, 4, and 6 h post MCAO. BQ788 (1 mg/kg, i.v.) wasadministered 15 min prior to the first injection of IRL-1620 or vehicle.Values are expressed as mean ± SEM (n = 5-8/group). Neurological RotaRod Distance Treatment Evaluation Grip Test Foot Fault Duration TraveledVertical Groups (6 point scale) (6 point scale) Error (%) (sec) (cm)Breaks Sham Baseline   0 ± 0 4.00 ± 0.29  3.96 ± 0.82  88.89 ± 9.18 4968± 242 65.62 ± 3.18 Day 1   0 ± 0 4.00 ± 0.29  4.04 ± 1.02 144.89 ± 9.233325 ± 324 37.63 ± 3.33 Day 4   0 ± 0 3.67 ± 0.24  4.57 ± 0.91 150.67 ±8.80 5323 ± 474 58.67 ± 1.53 Day 7   0 ± 0 4.00 ± 0.41  7.04 ± 2.50136.67 ± 19.61 4306 ± 314 53.67 ± 12.31 MCAO + Baseline   0 ± 0 3.89 ±0.26  4.56 ± 0.89 100.33 ± 7.54 5069 ± 329 54.56 ± 7.65 Vehicle Day 13.11 ± 0.31* 1.00 ± 0.29* 57.64 ± 7.39*  24.33 ± 5.87*  764 ± 216*  1.33± 0.53* Day 4 2.75 ± 0.57* 1.25 ± 0.32* 72.74 ± 6.63*  32.50 ± 13.57*2353 ± 787* 15.00 ± 7.53* Day 7 2.75 ± 0.57* 1.50 ± 0.43* 58.19 ± 10.85* 50.00 ± 18.55 2366 ± 660 20.25 ± 7.80* MCAO + Baseline   0 ± 0 3.86 ±0.46  4.05 ± 1.31 113.29 ± 8.34 5073 ± 334 61.50 ± 4.94 IRL-1620 Day 11.29 ± 0.36^(#) 2.71 ± 0.52^(#) 18.85 ± 6.48^(#)  78.71 ± 22.59* 1611 ±325* 12.75 ± 4.76* Day 4 0.67 ± 0.22^(#) 2.33 ± 0.58 14.00 ± 3.66^(#) 97.33 ± 2.08 2898 ± 451 26.33 ± 2.08 Day 7 0.67 ± 0.22^(#) 3.33 ± 0.22 8.28 ± 1.09^(#) 123.67 ± 7.28 3472 ± 732 34.33 ± 3.78 MCAO + Baseline  0 ± 0 3.33 ± 0.33  5.96 ± 1.75 102.00 ± 5.14 5141 ± 285 55.17 ± 3.74BQ788 Day 1 3.00 ± 0.58*^(@) 0.67 ± 0.33*^(@) 52.12 ± 11.93*^(@)  37.83± 20.87* 1168 ± 417*  5.33 ± 2.47* Day 4 3.33 ± 0.62*^(@) 0.33 ± 0.24*68.89 ± 12.27*^(@)  37.67 ± 14.92* 1246 ± 410*  4.33 ± 1.52* Day 7 3.00± 0.82*^(@) 1.67 ± 0.62 75.00 ± 17.67*^(@)  49.67 ± 17.56 2280 ± 83611.00 ± 3.97* MCAO + Baseline   0 ± 0 4.50 ± 0.34  4.92 ± 1.50 127.33 ±16.77 5642 ± 358 48.00 ± 9.68 BQ788 + Day 1 3.00 ± 0.37*^(@) 1.67 ±0.33* 61.01 ± 10.82*^(@)  51.17 ± 19.11*  742 ± 85*  2.00 ± 1.48*IRL-1620 Day 4 3.67 ± 0.47*^(@) 1.00 ± 0.40* 60.26 ± 14.59*^(@)  62.00 ±23.25 1223 ± 414  6.67 ± 3.42* Day 7 3.00 ± 0.82*^(@) 1.33 ± 0.62* 57.63± 17.63*^(@)  54.00 ± 35.04 2185 ± 818 17.33 ± 8.56* *P < 0.05 vs. sham.^(#)P < 0.05 vs. MCAO + vehicle. ^(@)P < 0.05 vs. MCAO + IRL-1620.

1. A method of treating a neuropsychiatric disorder comprisingadministering to a patient in need thereof a therapeutically effectiveamount of an endothelin-B receptor agonist to treat the neuropsychiatricdisorder.
 2. The method of claim 1 wherein the endothelin-B receptoragonist is co-administered with an additional agent to treat theneuropsychiatric disorder.
 3. The method of claim 2, wherein theadditional agent is selected from the group consisting of anantidepressant, an anti-inflammatory agent, a CNS stimulant, aneuroleptic, and an anti-proliferative agent.
 4. The method of claim 1wherein the endothelin-B receptor agonist is selected from the groupconsisting of IRL-1620, BQ-3020, [Ala^(1,3,11,15)]-Endothelin,Sarafotoxin S6c, endothelin-3, and a mixture thereof.
 5. The method ofclaim 1 wherein the neuropsychiatric disorder is selected from the groupconsisting of a cerebrovascular disease, stroke, cerebral ischemia,cerebral hemorrhage, head trauma, brain injury, a brain tumor, multiplesclerosis and demyelinating diseases, dementia, vascular dementia,Alzheimer's disease, Parkinson's disease, Huntington's disease, ataxia,motor neuron disease, Amyotrophic lateral sclerosis, drug intoxication,alcoholism, chronic brain infections, brain abscess, white matterdisease, Binswanger's disease, Moyamoya disease, perinatal hypoxia,cerebral asphyxia, intracranial birth injury, congenital malformation ofthe brain, mood disorders, and depression.
 6. The method of claim 1wherein the endothelin-B receptor agonist is administered at a doseranging from 0.0001 to 0.5 mg/kg.
 7. The method of claim 1 wherein theendothelin-B receptor agonist is administered repeatedly at intervals of1 to 6 hours after every two to five days.
 8. A composition comprising(a) an endothelin-B receptor agonist, (b) an agent used to treat aneuropsychiatric disorder, and optionally (c) an excipient.
 9. Anarticle of manufacture comprising: (a) a packaged composition comprisingan endothelin-b receptor agonist and an agent for treating aneuropsychiatric disorder; (b) an insert providing instructions for asimultaneous or sequential administration of the endothelin-b receptoragonist and the agent for the neuropsychiatric disorder to treat apatient; and (c) a container for (a) and (b).
 10. The method of claim 1,wherein the endothelin-B receptor agonist is IRL-1620.